Polyphenol proteasome inhibitors, synthesis, and methods of use

ABSTRACT

The present invention relates to synthetic green tea derived polyphenolic compounds, their modes of syntheses, and their use in inhibiting proteasomal activity and in treating cancers. The present invention is also directed to pharmaceutical compositions useful in methods of inhibiting proteasomes and of treating cancers.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.11/820,799, filed Jun. 20, 2007, which is a continuation of U.S.application Ser. No. 10/764,728, filed Jan. 26, 2004, now U.S. Pat. No.7,358,383, which claims benefit of U.S. Provisional Application Ser. No.60/442,213, filed Jan. 24, 2003, and U.S. Provisional Application Ser.No. 60/443,554, filed Jan. 30, 2003, which are hereby incorporated byreference herein in their entirety, including any figures, tables,nucleic acid sequences, amino acid sequences, or drawings.

BACKGROUND OF INVENTION

The proteasome is a massive multi-catalytic protease complex that isresponsible for degrading the majority of cellular proteins. The20S-core particle of the 26S proteasome is barrel-shaped, and the sitesof proteolytic activity reside on the interior.

The eukaryotic proteasome contains three known activities that areassociated with its β subunits. These are the chymotrypsin-like(cleavage after hydrophobic residues, β5 subunit), trypsin-like(cleavage after basic residues, β2 subunit), and caspase-like (cleavageafter acidic residues, β1 subunit) activities.

These three activities depend on the presence of an N-terminal Thr(Thr 1) residue. The hydroxyl group on the side chain of Thr 1 isresponsible for catalyzing cleavage of peptides through nucleophilicattack (addition-elimination mechanism). Near this N-terminal threonine,binding pockets recognize the side chains of peptides and give eachcatalytic site its specificity. The S1 pocket of the β5 subunit isdefined by the hydrophobic residues, Ala 20, Val 31, Ile 35, Met 45, Ala49, and Glu 53, and this binding pocket has been shown to be importantfor substrate specificity and binding of several types of proteasomeinhibitors.

The ubiquitin/proteasome-dependent degradation pathway plays anessential role in upregulation of cell proliferation, down-regulation ofcell death, and development of drug resistance in human tumor cells,suggesting the use of proteasome inhibitors as potential novelanticancer drugs, which has been demonstrated in various cell cultures,animal models and clinical trials. In a broad range of cell culturemodels, proteasome inhibitors rapidly induce tumor cell apoptosis,selectively trigger programmed cell death in the oncogene-transformed,but not normal or untransformed cells, and are able to activate thedeath program in human cancer cells that are resistant to variousanticancer agents. Inhibition of the chymotrypsin-like, but nottrypsin-like, activity has been found to be associated with induction oftumor cell apoptosis.

The proteasome degrades a number of proteins that are involved in tumorsuppression. Cyclin-dependent kinase inhibitor p27, a key regulatorymolecule in cell cycle progression, is one example (Pagano, M. et al.Science, 1995, 269:682-685). Inhibition of the proteasome results in anaccumulation of ubiquitinated and unmodified p27′ that can result in G₁cell cycle arrest (An, B. et al. Cell Death Differ, 1998, 5:1062-75;Sun, J. et al. Cancer Res, 2001, 61:1280-1284). Additionally, inhibitionof the proteasome increases the intracellular concentrations of IκB-a(Palombella, V. J. et al. Cell, 1994, 78:773-785), an inhibitor ofnuclear factor kappa B (NFκB), leading to inhibition of NFκB activation(Thompson, J. E. et al. Cell, 1995, 80:573-582) and reduction ofanti-apoptotic gene signaling (Perkins, N. D. Trends Biochem Sci, 2000,25:434-440). Another effect of proteasome inhibition is the accumulationof mitochondrial proapoptotic protein Bax, a Bc1-2 family member (Chang,Y. C. et al. Cell Growth Differ, 1998, 9:79-84; Li, B. and Dou, Q. P.Proc Natl Acad Sci USA, 2000, 97:3850-3855; Nam, S. et al. CancerEpidemiol Biomarkers Prey, 2001, 10:1083-1088), resulting in the releaseof cytochrome c from the mitochondria and activation of caspase-mediatedapoptosis (Green, D. R. and Reed, J. C. Science, 1998, 281:1309-1312).

In different animal studies, proteasome inhibitors suppress tumor growthvia induction of apoptosis and inhibition of angiogenesis. MLN-341(formerly PS-341) is a potent and selective dipeptidyl boronic acidcompound, which inhibits the chymotrypsin-like activity of the 20Sproteasome. This proteasome inhibitor is currently being developed forthe potential treatment of human hematological malignant neoplasms andsolid tumors. Preliminary data from Phase I and II clinical trialsconfirm the anti-tumor activity of MLN-341 although some associated sideeffects were observed. The proteasome inhibition mechanism of MLN-341has not been confirmed by X-ray diffraction experiments.

However, the proteasome-inhibition mechanism of another peptideinhibitor, LLnL, and nonpeptide inhibitors, such as lactacystin and themacrocyclic compound TMC-95, have been confirmed by X-ray diffraction.Understanding how these inhibitors function at the molecular level willgive insight into the structural studies of other proteasome inhibitorswhere X-ray crystal structures are not available. These studies therebydemonstrate that the proteasome is an excellent target for developingpharmacological anti-cancer drugs.

Tea, the most popular beverage in the world, is consumed by overtwo-thirds of the world's population. Several epidemiological studieshave provided evidence for the cancer-preventive properties of greentea. Furthermore, animal studies have also suggested that green teapolyphenols could suppress the formation and growth of various tumors.Although numerous cancer-related proteins are affected by teapolyphenols, the molecular basis for tea-mediated cancer preventionremains unknown.

The naturally occurring ester bond-containing green tea polyphenols(GTPs), such as (−)-epigallocatechin-3-gallate (also referred to hereinas (−)-EGCG, and shown in FIG. 1), possess the ability to inhibitproteasome activity both in vitro and in vivo. Recently completed PhaseI clinical trials using (−)-EGCG and green tea to treat cancer andprevent reoccurrence indicate a wide tolerance to green tea (up to 7-8cups/per day) (Pisters, K. M. et al. J Clin Oncol, 2001, 19:1830-1838).The lack of toxicity to normal cells observed in clinical trials andeffectiveness of treatment confirm the results from the cell culturemodels (Adams, J. et al. Cancer Res, 1999, 59:2615-2622; Dou, Q. P. andLi, B. et al. Drug Resist Updat, 1999, 2:215-223; Almond, J. B. andCohen, G. M. Leukemia, 2002, 16:433-443; Kisselev, A. F. and Goldberg,A. L. Chem Biol, 2001, 8:739-758). In addition, synthetic GTPs with anester bond, such as (+)-EGCG (shown in FIG. 1), are also able topotently and selectively inhibit the chymotrypsin-like activity of theproteasome. It appears that a center of nucleophilic susceptibilityresides at the ester bond carbon in these polyphenols. This proposedmechanism of ester bond-based nucleophilic attack is similar to that oflactacystin-based inhibition. However, a need still exists for moreoptions in inhibiting proteasome activity.

BRIEF SUMMARY OF THE INVENTION

Synthetic green tea polyphenol compounds potently inhibit proteasomalchymotrypsin-like activity. One aspect of the present invention pertainsto synthetic green tea-derived polyphenolic compounds useful forinhibiting proteasomal chymotrypsin-like activity. Polyphenoliccompounds of the present invention include (−)-EGCG-amides,(+)-EGCG-amides, and pharmaceutically acceptably salts and analogs ofthese compounds. Other polyphenolic compounds of the present inventioninclude those represented by the structures (formulas) shown in FIG. 8.

Another aspect of the present invention provides methods of synthesizing(−)-EGCG-amides and (+)-EGCG-amides. Advantageously, the reagents andsteps of the method of the invention can be easily adjusted to producestereoisomers of the products, if desired.

Another aspect of the present invention is directed to methods of usingthe polyphenolic compounds of the invention. In one embodiment, a methodto inhibit proteasomal activity is disclosed. Advantageously,proteasomal inhibition can take place in vivo or in vitro. In yetanother embodiment, a method for treating cancers is provided.

Another aspect of the present invention provides for pharmaceuticalcompositions containing polyphenolic compounds of the invention andpharmaceutically acceptable carriers or diluents.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication, withcolor drawing(s), will be provided by the Office upon request andpayment of the necessary fee.

FIG. 1 shows structures of green tea polyphenols (GTPs) and synthetic(−)-EGCG and (+)-EGCG amides.

FIG. 2A shows a view of the docking solution of (−)-EGCG and 20Sproteasome. The dotted yellow line represents the distance from thehydroxyl of Thr 1 to the carbonyl carbon of (−)-EGCG.

FIG. 2B shows an overview of (−)-EGCG binding mode to the β₅ subunit.The color code is: red, oxygen; blue, nitrogen; gray, carbon; and white,hydrogen.

FIG. 2C shows (−)-EGCG as a slick model with a mesh surface, bound tothe β5 binding cleft (ribbon representation).

FIG. 2D shows eight potential hydrogen (H) bonds formed between (−)-EGCGand indicated amino acids, with the yellow dotted lines representingH-bonds and the numbers next to the lines representing f1-bond distancesin Angstroms. The color code is red: oxygen; blue: nitrogen; gray:carbon; and white: hydrogen.

FIG. 2E also shows eight potential hydrogen (H) bonds formed between(−)-EGCG and indicated amino acids, with the yellow dotted linesrepresenting H-bonds and the numbers next to the lines representingf1-bond distances in Angstroms. The color code is red: oxygen; blue:nitrogen; gray: carbon; and white: hydrogen.

FIG. 2F shows the S1 hydrophobic pocket layered with a transparentsurface and residues that interact hydrophobically along with distancesfrom the residue to the A-C rings in (−)-EGCG.

FIG. 3A shows the binding mode of (−)-EGCG.

FIG. 3B shows the binding mode of (+)-EGCG.

FIG. 3B-1 shows an overlap of (−)-EGCG and (+)-EGCG (green) bindingmodes.

FIG. 3C shows the binding mode of (−)-GCG.

FIG. 3D shows the binding mode of (+)-GCG.

FIG. 3E shows the binding mode of (−)-ECG.

FIG. 3F shows the binding mode of (−)-CG.

FIG. 3G shows a two-dimensional scheme for the binding mode of (−)-GCG.The dotted lines represent potential hydrogen bond formation and the S1pocket designation represents hydrophobic interactions.

FIG. 3H shows a two-dimensional scheme for the binding mode of (+)-GCG.

FIG. 4A shows the nucleophilic susceptibility of (−)-EGCG. The whitecenter signifies the highest area of nucleophilic susceptibility.

FIG. 4B shows the nucleophilic susceptibility of (−)-EGCG-Amide. Thewhite center signifies the highest area of nucleophilic susceptibility.

FIG. 4C shows the binding mode of (−)-EGCG-Amide.

FIG. 4D shows the binding mode of (+)-EGCG-Amide.

FIG. 4E shows the bound conformation of (−)-EGCG, (+)-EGCG,(−)-EGCG-amide and (+)-EGCG-amide.

FIG. 4F shows an overlap of all the eight bound ligands and a bottomview of the saddle-shaped binding pocket. The surfaces are colored byatom type.

FIG. 5 shows a plot of predicted binding free energy (kcal/mol) againstthe actual proteasome-inhibitory activity (kcal/mol). A regressionanalysis yielded 0.9893 for the best fit R² value.

FIG. 6 shows a bar graph comparing the percentage of chymotrypsinactivity when a proteasomal cell comes into contact with any of theagents listed. A minus sign indicates that no dialysis took place; aplus signs indicates that dialysis took place.

FIG. 7 shows a graph for the kinetics of (−)-EGCG-mediated proteasomeinhibition as discussed in Example 4.

FIG. 8 shows structures of the natural tea polyphenol (−)-EGCG and eightsynthetic analogs, (−)-ECGC-A, (+)-EGCG, (+)-EGCG-A, GTP-1, GTP-2,GTP-3, GTP-4 and GTP-5 and their one-half maximal inhibition values(IC50s) of 20S proteasomal activity.

FIG. 9A shows the accumulation of p27, IκB-a, and Bax proteins by(−)-EGCG and (−)-EGCG-amide.

FIG. 9B shows the accumulation of p27, IκB-a, and Bax proteins by(+)-EGCG and (+)-EGCG-amide.

FIG. 10A shows the accumulation of p27, IκB-a, Bax, and ubiquitinatedproteins by GTP-1 and GTP-3.

FIG. 10B shows a Western blot assay using specific antibodies to p27,IκB-a, Bax, and ubiquitinated proteins for GTP-4, GTP-5 and (−)-EGCG.

FIG. 11A shows a DNA histogram for a control.

FIG. 11B shows a DNA histogram for GTP-4.

FIG. 11C shows a DNA histogram for GTP-5.

FIG. 12A shows soft agar colony formation of prostate cancer cells inthe present of a control, GTp-1, GTP-2, GTP-3, and (−)-EGCG.

FIG. 12B shows a bar graph of the mean number of colonies present ineach treatment plate.

FIG. 13A shows the selective accumulation of p27 and IκB-a proteins inleukemic Jurkat T over non-transformed NK cells when treated by(−)-EGCG.

FIG. 13B shows the selective accumulation of p27 and IκB-a proteins inleukemic Jurkat T over non-transformed NK cells when treated by(−)-EGCG-amide.

FIG. 13C shows a Western blot assay using specific antibodies to p27,IκB-a, ubiquitin, and actin.

FIG. 14A shows the preferable accumulation of p27 by (−)-EGCG-A,(+)-EGCG, (+)-EGCG-A, and GTP-1 in the transformed over the normal humanfibroblasts at twelve hours.

FIG. 14B shows the preferable accumulation of p27 by (−)-EGCG-A,(+)-EGCG, (+)-EGCG-A, and GTP-1 in the transformed over the normal humanfibroblasts at twelve hours.

FIG. 15 shows structures of synthetic and natural greet tea polyphenols.

FIG. 16A shows a bar graph of the inhibition of proteasomal activity bysynthetic and natural GTPs.

FIG. 16B shows a graph of the decrease in proteasomal activity as theconcentration of (+)-EGCG and (−)-EGCG increases.

FIG. 16C shows a graph of the decrease in proteasomal activity as theconcentration of (+)-GCG increases.

FIG. 17A shows a bar graph of chymotrypsin activity in the presence ofnatural and synthetic GTPs.

FIG. 17B shows a bar graph of trypsin activity in the presence ofnatural and synthetic GTPs.

FIG. 17C shows a bar graph of calpain activity in the presence ofnatural and synthetic GTPs.

FIG. 18A shows the accumulation of p27 and IkB-a proteins in Jurkatcells.

FIG. 18B shows the accumulation of p27 and IkB-a proteins in LNCaPcells.

FIG. 19 shows that synthetic GTPs induce G1 arrest in prostate cancerLNCaP cells.

FIG. 20A shows Bax-associated cancer cell apoptosis in cells of twohuman prostate cancer cell lines, LNCaP and DU145. High Bax protein (21kDa) levels in LNCaP cells, and very low Bax expression in DU145 cellswere observed.

FIG. 20B shows results of a Western blot using antibodies specific toBax and actin, where LNCaP or DU-145 cells were previously treated for24 hours with either H₂O (C, for control) or 10 μM (−)-EGCG or (+)-EGCG.

FIG. 20C shows a bar graph of caspase 3 activity in LNCaP cells andDU145 cells determined by cell-free caspase-3 activity assay followingtreatment with (−)-EGCG and (+)-EGCG for the indicted hours.

FIG. 20D shows results of a Western blot using PARP-specific antibody,where LNCaP or DU-145 cells were previously treated for 24 hours witheither H₂O(C, for control) or 10 μM (−)-EGCG or (+)-EGCG. (+)-EGCG, aswell as (−)-EGCG, induced the apoptosis-specific PARP cleavage only inLNCaP, but not DU145 cells.

FIG. 21A shows colony growth in the presence of synthetic and naturalGTPs.

FIG. 21B shows a bar graph of the mean number of colonies grown in thepresence of synthetic and natural GTPs.

FIG. 22A shows a bar graph demonstrating the effect of dialysis onproteasome inhibition.

FIG. 22B SDS-post-treatment does not inhibit (−)-EGCG-mediatedproteasome inhibition.

DETAILED DISCLOSURE OF THE INVENTION

The present invention is directed to polyphenolic compounds useful forinhibiting proteasomal activity, methods of synthesis and use inproteasome inhibition and treating cancer, and pharmaceuticalcompositions. In particular, the polyphenolic compounds of the presentinvention inhibit the chymotrypsin-like activity of a proteasome's β₅subunit. The polyphenolic compounds of the present invention may besynthesized using methods disclosed herein.

One embodiment of the subject invention is directed to polyphenoliccompounds having a similar ring structure to green tea polyphenols. Moreparticularly, the compounds of the present invention possess an adequatenumber of substituents to the phenols or carbonyl oxygens to ensurefavorable binding of the compounds to the β₅ subunit's active site.Subsequently, these compounds are capable of attack by the N-terminalThreonine (Thr 1) via acylation.

Advantageously, the compounds of the present invention are anirreversible mechanism-based inhibitor of the chymotrypsin-like activityof 20S proteasome.

The nomenclature of FIG. 1, whereby the rings of (−)-EGCG are named A,B, C or G, is utilized throughout the text.

In accordance with another embodiment of the present invention, there isprovided a compound having structure I:

wherein R₁-R₈ are independently selected from the group consisting of H,alkyl, alkenyl, cycloalkyl, heterocycloalkyl, cycloalkenyl,heterocycloalkenyl, aryl, and acyl group, any of which may be optionallysubstituted; and R₉ is selected from the group consisting of H, alkylgroup, alkenyl, cycloalkyl, heterocycloalkyl, cylcoalkenyl,heterocycloalkenyl, acyl, and aryl, any of which may be optionallysubstituted.

Optionally, formula I can be drawn as four separate two-dimensionalFisher Projection Formulations to represent the B ring of I projectingout and behind the A and C rings and the G ring of I projecting out andbehind the A and C rings.

In a specific embodiment, R₁-R₈ is selected from the group consisting of—H, alkyl, and acyl, and R₉ is selected from the group consisting of —H,alkyl, and aryl.

In a preferred embodiment, the carbonyl of both (+)-EGCG-amide and(−)-EGCG-amide compounds is susceptible to a nucleophilic attack due tothe presence of the amide nitrogen. As shown in FIGS. 4A and 4B,molecular orbital calculations confirmed that the amide bond-carbonproduced an arbitrary value of 0.55 for nucleophilic susceptibility. Incontrast, the same carbon in (−)-EGCG has a value of 0.69.

Introduction of a nitrogen atom into EGCG, as in EGCG-amide, reducesbond flexibility. It is known that such an amide bond (or peptide bond)is less flexible than the ester bond and prefers the trans conformation.

Therefore, due to the decreased flexibility of the amide bond, the amidepolyphenols cannot adopt a saddle-shaped conformation that isenergetically favorable for binding. This causes a straightening out ofthe arch conformation (FIG. 4E), which does not allow the A-C rings tobind as deeply in the S1 pocket as compared to (−)-EGCG, thus pullingthe compound further out of the binding cleft. This consequently raisesthe binding free energy of both (−)-EGCG-amide and (+)-EGCG-amide (−9.63and −9.52 kcal/mol, respectively; FIGS. 4C and 4D). This binding modewith increased binding free energy agrees with the increase in the IC₅₀values of both amide compounds (FIG. 4) and may (along with theirreduced nucleophilic susceptibility) explain their decreased potencyrelative to the corresponding esters.

However, the amide analogs were still able to accumulate levels of theproteasome target protein p27 in breast cancer MCF-7 cells, with potencycomparable to that of (−)-EGCG.

Another aspect of the present invention provides a method for thepreparation of a compound of the present invention, comprising the stepof coupling a compound of formula II with an acid of formula III to forma fully protected gallate ester, wherein R₁-R₉ are independently —H,alkyl, alkenyl, cycloalkyl, heterocylcoalkyl, cycloalkenyl,heterocycloalkenyl, acyl, or aryl.

In a specific embodiment, R₁-R₈ are independently aryl, and R₉ is —H.

Preferably the acid of formula III is employed in the form of aderivative which is an acyl halide or a mixed or symmetric acidanhydride, more preferably the derivative is an acyl halide, or the acidof formula III is reacted with the compound of formula II in thepresence of a condensing reagent.

In a preferred method of the present invention, the acid of formula IIIis reacted with a compound of formula II in the presence of a condensingagent, wherein said condensing agent is selected from the groupconsisting of 1,3-diisopropylcarbodiimide (DIPC),1,(3-dimethylaminopropyl(3-ethyl carbodiimide (EDC), dialkylcarbodiimide, 2-halo-1-alkyl-pyridinium halides, propane phosphonic acidcyclic anhydride (PPACA), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline(EEDQ) and dicyclohexylcarbodiimide (DCC).

Another embodiment of the present invention pertains to polyphenoliccompounds having double amide and ester bonds, such as compounds havingthe structures of FIG. 8 and labeled GTP-1, GTP-2, GTP-3, GTP-4 andGTP-5.

Although the compounds of the present invention can be administeredalone, one embodiment of the present invention is a pharmaceuticalformulation comprising at least one additional active ingredient,together with one or more pharmaceutically acceptable carrierstherefore. Each carrier must be acceptable in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient.

Pharmaceutical compositions are useful for inhibiting chymotrypsin-likeactivity of 20S proteasome, 26S proteasome, treating various cancers,for inhibiting cancer cell growth, for increasing the proportion of G₁cells in a population of cells (i.e., those cells occupying the G₁ phaseof the mitotic cell cycle), and for inducing apoptosis of cells. Onesuch composition comprises a compound selected from the group consistingof an enantiomer of an ester bond-containing tea polyphenol, an amideanalog of an ester bond-containing tea polyphenol, and an amide analogof an enantiomer of an ester bond-containing tea polyphenol, inassociation with a pharmaceutically acceptable carrier.

In a preferred composition according to an embodiment of the presentinvention, the compound has less than 100% optical purity.

In a preferred composition, the tea polyphenol is selected from thegroup consisting of the compounds of the present invention, morepreferably (+)-EGCG-amide, (−)-EGCG-amide, any of the compounds whosestructures are listed in FIG. 8, pharmaceutically acceptable salts,analogs, and mixtures thereof.

Another preferred composition utilizes polyphenols having the structuresof GTP-1, GTP-2, GTP-3, GTP-4 and GTP-5 (FIG. 8), pharmaceuticallyacceptable salts, analogs, and mixtures thereof.

Formulations include those suitable for oral, rectal, nasal, topical(including transdermal, buccal and sublingual), vaginal, parental(including subcutaneous, intramuscular, intravenous and intradermal) andpulmonary administration. The formulations can conveniently be presentedin unit dosage form and can be prepared by any methods well known in theart of pharmacy. Such methods include the step of bringing intoassociation the active ingredient with the carrier which constitutes oneor more accessory ingredients. In general, the formulations are preparedby uniformly and intimately bringing into association the activeingredient with liquid carriers or finely divided solid carriers orboth, and then if necessary shaping the product. Formulations of thepresent invention suitable for oral administration can be presented asdiscrete units such as capsules, cachets or tablets, each containing apredetermined amount of the active ingredient; or as an oil-in-waterliquid emulsion, water-in-oil liquid emulsion or as a supplement withinan aqueous solution, for example, a tea. The active ingredient can alsobe presented as bolus, electuary, or paste.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Pharmaceutical compositions for topical administration according to thepresent invention can be formulated as an ointment, cream, suspension,lotion, powder, solution, paste, gel, spray, aerosol or oil.Alternatively, a formulation can comprise a patch or a dressing such asa bandage or adhesive plaster impregnated with active ingredients, andoptionally one or more excipients or diluents.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the agent.

Formulations for rectal administration can be presented as a suppositorywith a suitable base comprising, for example, cocoa butter or asalicylate.

Formulation suitable for vaginal administration can be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the agent, such carriers as are known in theart to be appropriate.

Formulations suitable for nasal administration, wherein the carrier is asolid, include a coarse powder having a particle size, for example, inthe range of about 20 to about 500 microns which is administered in themanner in which snuff is taken, i.e., by rapid inhalation through thenasal passage from a container of the powder held close up to the nose.Suitable formulations wherein the carrier is a liquid for administrationby nebulizer, include aqueous or oily solutions of the agent.

Formulations suitable for parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions which can containantioxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which can include suspendingagents and thickening agents, and liposomes or other microparticulatesystems which are designed to target the compound to blood components orone or more organs. The formulations can be presented in unit-dose ormulti-does or multi-dose sealed containers, such as for example,ampoules and vials, and can be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample water for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions can be prepared from sterilepowders, granules and tablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose orunit, daily subdose, as herein above-recited, or an appropriate fractionthereof, of an agent. It should be understood that in addition to theingredients particularly mentioned above, the formulations of thisinvention can include other agents conventional in the art regarding thetype of formulation in question. For example, formulations suitable fororal administration can include such further agents as sweeteners,thickeners, and flavoring agents. It also is intended that the agents,compositions, and methods of this invention be combined with othersuitable compositions and therapies.

Various delivery systems are known and can be used to administer atherapeutic agent of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, receptor-mediated endocytosis and thelike. Methods of delivery include, but are not limited to,intra-arterial, intramuscular, intravenous, intranasal, and oral routes.In a specific embodiment, the pharmaceutical compositions of theinvention can be administered locally to the area in need of treatment;such local administration can be achieved, for example, by localinfusion during surgery, by injection, or by means of a catheter.

Therapeutic amounts can be empirically determined and will vary with thepathology being treated, the subject being treated, and the efficacy andtoxicity of the agent. Similarly, suitable dosage formulations andmethods of administering the agents can be readily determined by thoseof skill in the art.

The pharmaceutical compositions can be administered by any of a varietyof routes, such as orally, intranasally, parenterally or by inhalationtherapy, and can take form of tablets, lozenges, granules, capsules,pills, ampoule, suppositories or aerosol form. They can also take theform of suspensions, solutions, and emulsions of the active ingredientin aqueous or nonaqueous diluents, syrups, granulates or powders. Inaddition to an agent of the present invention, the pharmaceuticalcompositions can also contain other pharmaceutically active compounds ora plurality of compounds of the invention.

Ideally, the agent should be administered to achieve peak concentrationsof the active compound at sites of the disease. Peak concentrations atdisease sites can be achieved, for example, by intravenously injectingof the agent, optionally in saline, or orally administering, example, atablet, capsule or syrup containing the active ingredient.

Advantageously, the compositions can be administered simultaneously orsequentially with other drugs or biologically active agents. Examplesinclude, but are not limited to, antioxidants, free radical scavengingagents, peptides, growth factors, antibiotics, bacteriostatic agents,immunosuppressives, anticoagulants, buffering agents, anti-inflammatoryagents, anti-pyretics, time-release binders, anesthetics, steroids andcorticosteroids.

Another aspect of the present invention is directed to methods ofinhibiting proteasomal activity. In particular, the chymotrypsinactivity and chymotrypsin-like activity of the 20S proteasome isinhibited.

One method of inhibiting comprises contacting a proteasomal cell with asufficient amount of the compounds of the present invention.Advantageously, inhibition can take place in vivo or in vitro.

Another embodiment of the present invention provides a method ofinhibiting chymotrypsin-like activity of 20S proteasome, comprisingadministering to an individual a pharmaceutically effective amount of apharmaceutical composition of the present invention.

Preferably, the administering is carried out orally, parenterally,subcutaneously, intravenously, intramuscularly, intraperitoneally,intraarterially, transdermally or via a mucus membrane.

In accordance with another embodiment of the present invention, there isprovided a method of treating cancer, comprising administering to anindividual a pharmaceutically effective amount of a pharmaceuticalcomposition of the present invention.

Preferably, a cancer to be treated in accordance with an embodiment ofthe present invention is selected from the group consisting of prostatecancer, leukemia, hormone dependent cancers, breast cancer, coloncancer, lung cancer, epidermal cancer, liver cancer, esophageal cancer,stomach cancer, cancer of the brain, and cancer of the kidney.

In a preferred method, the treatment is effected by inducing apoptosisof cells of the cancer.

Another embodiment of the present invention provides a method ofinhibiting cancer cell growth, comprising administering to a patient apharmaceutically effective amount of a pharmaceutical composition of thepresent invention.

In accordance with another embodiment of the present invention, there isprovided a method of increasing relative proportion of G₁ cells in apopulation of cells, such as cancer cells, comprising administering to apatient a pharmaceutically effective amount of a pharmaceuticalcomposition according to an embodiment of the present invention.

In accordance with another embodiment of the present invention, there isprovided a method of inducing apoptosis in a population of cells,preferably cancer cells, such as LNCaP cells, by administering to thepopulation of cells an effective amount of a composition according to anembodiment of the present invention.

For the purpose of the present invention. the following terms aredefined below.

The term “inhibition” is intended to mean a substantially slowing,interference, suppression, prevention, delay and/or arrest of a chemicalaction.

The term “pharmacological inhibition” is intended to mean asubstantially slowing, interference, suppression, prevention, delayand/or arrest of a chemical action which is caused by an effectiveamount of a compound, drug, or agent.

The term “inhibitor” is intended to mean a compound, drug, or agent thatsubstantially slows, interferes, suppresses, prevents, delays and/orarrests a chemical action.

The term “green tea” is intended to mean non-fermented leaves of the teaplant Camellia sinensis

The term “black tea” is intended to mean fermented leaves of the teaplant Camellia sinensis.

The term “polyphenol” is intended to mean a compound with more than onephenolic moiety. A phenolic compound is an aromatic compound containingan aromatic nucleus to which is directly bonded at least one hydroxylgroup. The term polyphenol includes, without limitation, (−)EGCG,(−)EGC, (−)ECG, and (−)EC, such as those that can be extracted fromleaves of the tea plant Camellia sinensis, and analogs thereof, as wellas structurally similar synthetic analogs.

The term “tea polyphenol” is intended to mean a polyphenol capable ofbeing extracted/isolated from leaves of the tea plant Camellia sinensis,but which may be chemically synthesized. The term green tea polyphenolspecifically means a polyphenol capable of being extracted from greentea leaves.

The term “alkyl group” is intended to mean a group of atoms derived froman alkane by the removal of one hydrogen atom. Thus, the term includesstraight or branched chain alkyl moieties including, for example,methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, hexyl, andthe like. Preferred alkyl groups contain from 1 to about 6 carbon atoms(C₁₋₆ alkyl).

The term “aryl group” is intended to mean a group derived from anaromatic hydrocarbon by removal of a hydrogen from the aromatic system.Preferred aryl groups contain phenyl or substituted phenyl groups. Thus,the term “aryl” includes an aromatic carbocyclic radical having a singlering or two condensed rings. This term includes, for example, phenyl ornaphthyl.

The term “heteroaryl” refers to aromatic ring systems of five or moreatoms (e.g., five to ten atoms) of which at least one atom is selectedfrom O, N and S, and includes for example furanyl, thiophenyl, pyridyl,indolyl, quinolyl and the like.

The term “acyl group” is intended to mean a group having the formulaRCO—, wherein R is an alkyl group or an aryl group.

The term “alkenyl” refers to a straight or branched chain alkyl moietyhaving two or more carbon atoms (e.g., two to six carbon atoms, C₂₋₆alkenyl) and having in addition one double bond, of either E or Zstereochemistry where applicable. This term would include, for example,vinyl, 1-propenyl, 1- and 2-butenyl, 2-methyl-2-propenyl, etc.

The term “cycloalkyl” refers to a saturated alicyclic moiety havingthree or more carbon atoms (e.g., from three to six carbon atoms) andwhich may be optionally benzofused at any available position. This termincludes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,indanyl and tetrahydronaphthyl.

The term “heterocycloalkyl” refers to a saturated heterocyclic moietyhaving three or more carbon atoms (e.g., from three to six carbon atoms)and one or more heteroatom from the group N, O, S (or oxidised versionsthereof) and which may be optionally benzofused at any availableposition. This term includes, for example, azetidinyl, pyrrolidinyl,tetrahydrofuranyl, piperidinyl, indolinyl and tetrahydroquinolinyl.

The term “cycloalkenyl” refers to an alicyclic moiety having three ormore carbon atoms (e.g., from three to six carbon atoms) and having inaddition one double bond. This term includes, for example, cyclopentenylor cyclohexenyl.

The term “heterocycloalkenyl” refers to an alicyclic moiety having fromthree to six carbon atoms and one or more heteroatoms from the group N,O, S (or oxides thereof) and having in addition one double bond. Thisterm includes, for example, dihydropyranyl.

The term “halogen” means a halogen of the periodic table, such asfluorine, chlorine, bromine, or iodine.

The term “optionally substituted” means optionally substituted with oneor more of the aforementioned groups (e.g., alkyl, aryl, heteroaryl,acyl, alkenyl, cycloalkyl, heterocycloalkyl, cycloalkenyl,heterocycloalkenyl, or halogen), at any available position or positions.

The term “extracted” is intended to mean that the compound is isolatedfrom all or at least some of the components that accompany it in nature.The term “isolated” is inclusive of “extracted” and is intended to meanthat the compound is isolated from all or at least some of thecomponents that accompany it in nature or in its synthesis. For example,according to methods of the present invention, the polyphenoliccompounds can be administered or contacted to cells in vivo or in vitro,wherein the compound is in an isolated or non-isolated form, and with orwithout carriers, diluents, or additional agents.

The term “isomer” is intended to mean a compound that has the samemolecular formula as another compound. All isomers fall into either oftwo groups: structural isomers or stereoisomers.

The term “stereoisomer” is intended to mean a compound that has itsatoms joined in the same order as another compound but differs in theway its atoms are arranged in space. Stereoisomers can be subdividedinto two categories: enantiomers and diastereomers.

The term “enantiomer” is intended to mean a stereoisomer that is relatedlike an object and its mirror reflection. Enantiomers occur only withcompounds whose molecules are chiral, that is, with molecules that arenot superposable on their mirror reflections. Separate enantiomersrotate the plane of polarized light and are said to be optically active.They have equal but opposite specific rotations.

The term “racemic” is intended to mean an equimolar mixture ofenantiomers.

The term “optical purity” is intended to mean an indication of thepurity of a single enantiomer in an optically active substance. A sampleof an optically active substance that consists of a single enantiomer issaid to be 100% optically pure. An optically active substance thatcontains less than 100% optical purity contains more than a singleenantiomer.

The term “analog” is intended to mean a compound that is similar orcomparable, but not identical, to a reference compound, i.e. a compoundsimilar in function and appearance, but not in structure or origin tothe reference compound. For example, the reference compound can be areference green tea polyphenol and an analog is a substance possessing achemical structure or chemical properties similar to those of thereference green tea polyphenol. As used herein, an analog is a chemicalcompound that may be structurally similar to another but differs incomposition (as in the replacement of one atom by an atom of a differentelement or in the presence of a particular functional group). An analogmay be extracted from a natural source or be prepared using syntheticmethods.

The term “cancer” is intended to mean any cellular malignancy whoseunique trait is the loss of normal controls which results in unregulatedgrowth, lack of differentiation and ability to invade local tissues andmetastasize. Cancer can develop in any tissue of any organ. Morespecifically, cancer is intended to include, without limitation,prostate cancer, leukemia, hormone dependent cancers, breast cancer,colon cancer, lung cancer, epidermal cancer, liver cancer, esophagealcancer, stomach cancer, cancer of the brain, and cancer of the kidney.

The terms “treatment”, “treating” and the like are intended to meanobtaining a desired pharmacologic and/or physiologic effect, e.g.,inhibition of cancer cell growth or induction of apoptosis of a cancercell. The effect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment” as used herein covers anytreatment of a disease in a mammal, particularly a human, and includes:(a) preventing a disease or condition (e.g., preventing cancer) fromoccurring in an individual who may be predisposed to the disease but hasnot yet been diagnosed as having it; (b) inhibiting the disease, (e.g.,arresting its development); or (c) relieving the disease (e.g., reducingsymptoms associated with the disease).

The term “anti-cancer activity” is intended to mean an activity which isable to substantially inhibit, slow, interfere, suppress, prevent, delayand/or arrest a cancer and/or a metastasis thereof (such as initiation,growth, spread, and/or progression thereof of such cancer and/ormetastasis).

The term “biological activity” is intended to mean having the ability toinhibit chymotrypsin-like activity of the proteasome. Biologicalactivity is also intended to mean having the ability to inhibit cellgrowth, induce apoptosis, and/or suppress the transforming activity incancer cells.

The term “natural compound” is intended to mean a compound extractablefrom a natural source.

The term “administering” and “administration” is intended to mean a modeof delivery including, without limitation, oral, rectal, parenteral,subcutaneous, intravenous, intramuscular, intraperitoneal,intraarterial, transdermally or via a mucus membrane. The preferred onebeing orally. One skilled in the art recognizes that suitable forms oforal formulation include, but are not limited to, a tablet, a pill, acapsule, a lozenge, a powder, a sustained release tablet, a liquid, aliquid suspension, a gel, a syrup, a slurry, a suspension, and the like.For example, a daily dosage can be divided into one, two or more dosesin a suitable form to be administered at one, two or more timesthroughout a time period.

The term “therapeutically effective” is intended to mean an amount of acompound sufficient to substantially improve some symptom associatedwith a disease or a medical condition. For example, in the treatment ofcancer, a compound which decreases, prevents, delays, suppresses, orarrests any symptom of the disease would be therapeutically effective. Atherapeutically effective amount of a compound is not required to cure adisease but will provide a treatment for a disease such that the onsetof the disease is delayed, hindered, or prevented, or the diseasesymptoms are ameliorated, or the term of the disease is changed or, forexample, is less severe or recovery is accelerated in an individual.

The term “chymotrypsin-like activity” refers to the ability of theeukaryotic proteasome β subunit to cleave amino acid sequences afterhydrophobic residues, and is intended to include chymptrypsin activity.

The polyphenolic compounds of the present invention may be used incombination with either conventional methods of treatment and/or therapyor may be used separately from conventional methods of treatment and/ortherapy.

When the compounds of this invention are administered in combinationtherapies with other agents, they may be administered sequentially orconcurrently to an individual. Alternatively, pharmaceuticalcompositions according to the present invention may be comprised of acombination of a compound of the present invention, as described herein,and another therapeutic or prophylactic agent known in the art.

It will be understood that a specific “effective amount” for anyparticular in vivo or in vitro application will depend upon a variety offactors including the activity of the specific compound employed, theage, body weight, general health, sex, and/or diet of the individual,time of administration, route of administration, rate of excretion, drugcombination and the severity of the particular disease undergoingprevention or therapy. For example, the “effective amount” may be theamount of polyphenol compound of the invention necessary to achieveinhibition (e.g., diminishment or abatement) of proteosomalchymotrypsin-like activity in vivo or in vitro. The “effective amount”may be the amount of polyphenol compound of the invention necessary toachieve apoptosis or an increase in the relative number of G₁ cells.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includecitric acid, lactic acid, tartaric acid, fatty acids, and the like.

Salts may also be formed with bases. Such salts include salts derivedfrom inorganic or organic bases, for example alkali metal salts such asmagnesium or calcium salts, and organic amine salts such as morpholine,piperidine, dimethylamine or diethylamine salts.

As used herein, the term “pharmaceutically acceptable carrier” includesany and all solvents (such as phosphate buffered saline buffers, water,saline), dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions. The pharmaceutical compositionsof the subject invention can be formulated according to known methodsfor preparing pharmaceutically useful compositions. Formulations aredescribed in a number of sources which are well known and readilyavailable to those skilled in the art. For example, Remington'sPharmaceutical Science (Martin E W (1995) Easton Pa., Mack PublishingCompany, 19^(th) ed.) describes formulations which can be used inconnection with the subject invention.

As used herein, the terms “individual” and “patient” are usedinterchangeably to refer to any vertebrate species, such as humans andanimals. Preferably, the patient is of a mammalian species. Mammalianspecies which benefit from the disclosed methods of treatment include,and are not limited to, apes, chimpanzees, orangutans, humans, monkeys;domesticated animals (e.g., pets) such as dogs, cats, guinea pigs,hamsters, Vietnamese pot-bellied pigs, rabbits, and ferrets;domesticated farm animals such as cows, buffalo, bison, horses, donkey,swine, sheep, and goats; exotic animals typically found in zoos, such asbear, lions, tigers, panthers, elephants, hippopotamus, rhinoceros,giraffes, antelopes, sloth, gazelles, zebras, wildebeests, prairie dogs,koala bears, kangaroo, opossums, raccoons, pandas, hyena, seals, sealions, elephant seals, otters, porpoises, dolphins, and whales. Human ornon-human animal patients can range in age from neonates to elderly.

Materials and Methods

Materials. Highly purified tea polyphenols (−)-EGCG (>95%), (−)-GCG(>98%), (−)-ECG (>98%), (−)-CG (>98%), fetal calf serum, propidiumiodide, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide(MTT), RNase A, and DMSO were purchased from Sigma (St. Louis, Mo.).(+)-EGCG, benzyl-protected-(+)-EGCG, (+)-GCG, (−)-EGCG-amide, and(+)-EGCG-amide were prepared by enantioselective synthesis (see below).Purified 20S eukaryotic proteasome from rabbit was purchased from BOSTONBIOCHEM (Cambridge, Mass.). Purified 20S prokaryotic proteasome(Methanosarcina thermophile, Recombinant, E. coli) was purchased fromCALBIOCHEM (La Jolla, Calif.). Fluorogenic peptide substratesSuc-Leu-Leu-Val-Tyr-AMC (for the proteasomal chymotrypsin-like activity)was obtained from Calbiochem (La Jolla, Calif.). RPMI 1640 medium,Dulbecco's modified Eagle's medium, MEM non-essential amino acidssolution, MEM-sodium pyruvate solution, penicillin, and streptomycinwere from INVITROGEN (Carlsbad, Calif.). Monoclonal antibody top27^(Kip) was purchased from PHARMINGEN (San Diego, Calif.); polyclonalantibodies to IκB-a, Bax and actin, and monoclonal antibody to ubiquitinwere from SANTA CRUZ Biotechnology Inc. (Santa Cruz, Calif.).

Inhibition of purified 20S proteasome activity by natural or syntheticGTPs. Purified prokaryotic (0.5 μg) or eukaryotic (0.02 μg) 20Sproteasome was incubated with 20 μM fluorogenic peptide substrate,Suc-Leu-Leu-Val-Tyr-AMC for 30 mM at 37° C. in 100 μl of assay buffer(50 mM Tris-HCl, pH 7.5), with or without a natural or synthetic teapolyphenol. After incubation, production of hydrolyzed7-amido-4-methyl-coumarin (AMC) groups was measured using a multi-wellplate VERSAFLUOR Fluorometer with an excitation filter of 380 nm and anemission filter of 460 nm (BIO-RAD), and a multi-well plate readerWallac 1420 VICTOR™² with an excitation filter of 355 nm and an emissionfilter of 460 nm (EG&G WALLAC).

Assays for irreversible inhibition. To measure the effect of dialysis on(−)-EGCG-mediated proteasome inhibition, 20S prokaryotic proteasome (2μg) was incubated with 10 (−)-EGCG or the control solvent (H₂O) in 50 mMTris-HCl, pH 7.5 for 1 hour. This was then incubated at 4° C. eitherwithout or with dialysis overnight using a 10,000 MWCO PierceSlide-A-Lyzer Dialysis Cassette (Rockford, Ill.) in a rotating bath of50 mM Tris-HCl, pH 7.5. The proteasomal chymotrypsin-like activity wasthen assayed as previously described. As a control, an EGCG solution(without purified 20S proteasome) was dialyzed overnight, followed bymeasurement of the effects on inhibition of the proteasome activity.

Molecular modeling and docking studies. The crystal structure of theeukaryotic yeast 20S proteasome was obtained from the Protein Database(Ref. Number 1JD2), and used for the docking examples. The yeast 20Sproteasome is structurally very similar to the mammalian 20S proteasome,and the chymotrypsin active site between the two species is highlyconserved.

The AutoDock suite of programs, which was used for the dockingcalculations, employs an automated docking approach allowing ligandflexibility as described to a full extent elsewhere. AutoDock has beencompared to various docking programs in several studies and hasconsistently produced docked conformations that predict X-ray crystalstructures with bound ligands. Default parameters were used as isdescribed in the AutoDock manual except for those changes mentionedbelow. The dockings were run on an i386 architecture computer runningRedhat Linux 6.0.

The crystal structure of the 20S proteasome and the ligands wereprepared for docking by following the default protocols except werenoted. The energy-scoring grid was prepared as a 20×20×20 Å box centeredaround the β5 catalytic N-terminal threonine, and the ligand was limitedto this search space during docking. Atomic solvation parameters wereassigned to the proteasome using default parameters. The defaultparameters for the Lamarckian genetic algorithm were used as the searchprotocol except for the maximum number of energy evaluations, which werechanged to 5 million. This Lamarckian genetic algorithm method withAutoDock has previously been shown to reproduce binding modes verysimilar to crystal structures with bound ligands. AutoDock relies uponan empirical scoring function that provides approximate binding freeenergies. The default parameters were kept for mutation, crossover andelitism. The pseudo-Solis and Wets local search method was included asdefault parameters as well.

Docking modes were selected based on two criteria: their proximity tothe N-terminal threonine and placement of the ring system of themolecule within the S1 hydrophobic pocket. Of theorientations/conformations that fit these two criteria, the dockedstructure of lowest free energy was chosen. Each molecule was dockedwith up to 30 runs of 5 million energy evaluations for each run. Theoutput from AutoDock and all modeling studies as well as images wereproduced with PyMOL. PyMOL was used to calculate the distances ofhydrogen bonds as measured between the hydrogen and its respective atom.

Determination of nucleophilic susceptibility. The electron densitysurface colored by nucleophilic susceptibility was created by performingExtended Hückel molecular orbital calculations using Cache Worksystemver. 3.2 (Oxford Molecular Ltd., now Accelrys). A colored “bull's-eye”with a white-center denotes atoms that are highly susceptible tonucleophilic attack.

Cell Culture, Cell Extract Preparation, and Western Blot Assay. Humanleukemia Jurkat T and prostate cancer LNCaP cells were cultured in RPMI1640 medium supplemented with 10% fetal calf serum, 100 units/ml ofpenicillin, and 100 μg/ml streptomycin. Human immortalized,non-transformed NK cells (YT cell line) (38) were cultured in RPMI 1640supplemented with 1 mM MEM sodium pyruvate solution, 0.1 mM MEMnon-essential amino acids solution, 10% fetal calf serum. 100 units/mlpenicillin, and 100 μg/ml streptomycin. Normal (WI-38) and simian virus40 (SV40)-transformed (VA-13) human fibroblasts were grown in Dulbecco'smodified Eagle's medium supplemented with 10% fetal calf serum, 100units/ml of penicillin and 100 μg/ml of streptomycin. All cells weremaintained at 37° C. in a humidified incubator with an atmosphere of 5%CO₂. A whole cell extract was prepared in accordance with An et al. (An,B. et al. Cell Death Differ, 1998, 5:1062-75). Briefly, cells wereharvested, washed with PBS twice, and homogenized in a lysis buffer [50mM Tris-HCl (pH 8.0), 5 mM EDTA, 150 mM NaCl, 0.5% NP-40, 0.5 mMphenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol] for 30 min at4° C. Afterwards, lysates were centrifuged at 12,000×g for 15 min at 4°C., and the supernatants were collected as whole cell extracts. Equalamounts of protein extract (60 μg) were resolved by SDS-polyacrylamidegel electrophoresis and transferred to a nitrocellulose membrane using aSemi-Dry Transfer system (BIO-RAD, Hercules, Calif.). Jurkat T, LNCaP,or DU-145 cells were treated with various concentrations of synthetic ornatural tea polyphenols for indicted hours. The enhancedchemiluminescence (ECL) Western blot analysis was then performed usingspecific antibodies to the proteins of interest (p²⁷kip, IκB-a, PARP,Bax or actin). The ratio of p27 or IκB-α to the corresponding actin wasdetermined by scanning intensities of the protein bands.

Flow Cytometry. Cell cycle analysis based on DNA content was performedas in An et al. (An, B. et al. Cell Death Differ, 1998, 5:1062-75). Ateach time point, cells were harvested, counted, and washed twice withPBS. Cells (5×10⁶) were suspended in 0.5 ml PBS, fixed in 5 ml of 70%ethanol overnight at −20° C., centrifuged, resuspended again in 1 ml ofpropidium iodide staining solution (50 μg propidium iodide, 100 unitsRNase A and 1 mg glucose per ml PBS), and incubated at room temperaturefor 30 minutes. The cells were then analyzed with FACScan (BectonDickinson Immunocytometry, CA), ModFit LT and WinMDI V.2.8 cell cycleanalysis software (Verity Software; Topsham, ME). The cell cycledistribution is shown as the percentage of cells containing G₁, S, G₂,and M phase DNA judged by propidium iodide staining. The apoptoticpopulation is determined as the percentage of cells with sub-G₁ DNAcontent (<G₁).

Soft Agar Assay. The soft agar assay was performed according to Smith etal. (Smith, D. M. et al. Mol Med, 2002, 8:382-392) with a fewmodifications. In brief, in a six-well plate, a bottom feeder layer(0.6% agar) was prepared with DMEM media containing 10% fetal bovineserum, 100 units/ml penicillin, and 100 μg/ml streptomycin. A top layer(0.3% agar) was prepared with DMEM and the same media as described abovebut containing 2×10⁴ prostate cancer LNCaP cells and indicated analogsor DMSO as a control. Plates were incubated at 37° C. in 5% CO₂ in ahumidified incubator for two weeks. MTT (1 mg/ml) was added to each welland incubated overnight to allow complete formation of purple formazancrystals. The plates were then scanned and photographed, and the numberof colonies was quantified by QUANTITY ONE v.4.0.3 software (Bio-Rad,Hercules, Calif.).

Example 1 (+)-EGCG-amide and (−)-EGCG-amide Potency for ProteasomalInhibition

Purified prokaryotic (0.5 μg) or eukaryotic (0.02 μg) 20S proteasome wasincubated with 20 μM fluorogenic peptide substrate,Suc-Leu-Leu-Val-Tyr-AMC for 30 minutes at 37° C. in 100 μl of assaybuffer (50 nM Tris-HCl, pH 7.5), with or without a natural or synthetictea polyphenol. After incubation, production of hydrolyzed AMC groupswas measured using a multi-well plate VERSAFLUOR Fluorometer with anexcitation filter of 380 nm and an emission filter of 460 nm.

The IC₅₀ values against 20S eukaryotic proteasome were determined to be320 and 405 nM for both (−)-EGCG-amide and (+)-EGCG-amide, respectively(FIGS. 4C and 4D). Compared to (−)- and (+)-EGCG, both amide compoundshave decreased proteasome-inhibitory potencies respectively (FIGS. 4Cand 4D vs. FIGS. 3A and 3B) although their stereochemical structureswere not changed (FIG. 1).

Example 2 Inhibition of the Proteasomal Activity by SyntheticEGCG-Amides in Intact Prostate Cancer Cells

To investigate whether (−)-EGCG-amide and (+)-EGCG-amide inhibit intacttumor cell proteasome activity, human prostate cancer LNCaP cells weretreated with 25 μM of (−)- or (+)-EGCG-amide for up to 9 hours, followedby measurement of p27, IκB-a and Bax, three well-known proteasome targetproteins (Pagano, M. et al. Science, 1995, 269:682-685; An, B. et al.Cell Death Differ, 1998, 5:1062-75; Sun, J. et al. Cancer Res, 2001,61:1280-1284; Palombella, V. J. et al. Cell, 1994, 78:773-785; Thompson,J. E. et al. Cell, 1995, 80:573-582; Perkins, N. D. Trends Biochem Sci,2000, 25:434-440; Chang, Y. C. et al. Cell Growth Differ, 1998, 9:79-84;Li, B. and Dou, Q. P. Proc Natl Acad Sci USA, 2000, 97:3850-3855; Nam,S. et al. Cancer Epidemiol Biomarkers Prey, 2001, 10:1083-1088). In thisexperiment (−)-EGCG and (+)-EGCG were used as positive controls. Similarto (−)-EGCG and (+)-EGCG, the two corresponding EGCG amides were able toincrease levels of p27, IκB-a and Bax by 3 hours, which remained high upto 9 hours (FIGS. 9A-9B). Both (−)- and (+)-EGCG-amide were also able toinhibit the proteasome activity in human breast cancer MCF-7 cells. Thisdemonstrates that EGCG amides are capable of inhibiting the proteasomechymotrypsin-like activity in intact tumor cells.

Example 3 Synthetic EGCG Analogs with Double Ester or Amide BondsInhibit the Chymotrypsin-Like Activity of Purified 205 Proteasome

To further study the functional roles of the ester or amide bond of EGCGcompounds in inhibiting the proteasomal activity, several GTP analogscontaining an additional ester bond between B- and C-rings weredesigned. To overcome the stability problem, the C-ring oxygen wasreplaced by the isosteric CH₂ group (FIG. 8). These compounds, namedGTP-1 to −5 (FIG. 8), were synthesized and tested forproteasome-inhibitory activity.

Potencies of three analogs without the A-ring, GTP-1, -2 and -3 wereinvestigated first. GTP-1 with cis-diesters has an IC₅₀ value of 1.9 μMagainst the chymotrypsin-like activity of purified 20S proteasome (FIG.8). GTP-2 containing cis-diamides has an IC₅₀ value of 2.5 μM, which isslightly less potent than GTP-1. GTP-3 with trans-diamides has an IC₅₀value of 1.5 μM (FIG. 8). The decreased potencies of GTP-1, -2 and -3,compared to that of the natural (−)-EGCG (IC₅₀ 0.1-0.2 μM; 17, 36),suggest that the A-ring is required for inhibiting the proteasomeactivity.

To further examine the A-ring requirement, the potencies of the two“A-C-ring” analogs, GTP-4 and GTP-5, was measured. GTP-4 containscis-diesters and has IC₅₀ of 0.34 μM (FIG. 8). Addition of a hydroxylgroup to the A-ring of GTP-4 generates GTP-5 (FIG. 8), whose IC₅₀ wasfound to be 0.73 μM. Compared to GTP-1, -2 and -3, GTP-4 and -5compounds have increased potencies, consistence with the requirement ofthe A-ring for proteasome inhibition.

Example 4 Accumulation of Proteasome Target and PolyubiquitinatedProteins and Induction of G₁ Arrest and Cell Death by GTP Analogs

Having determined that the synthetic GTP-1 to −5 analogs are able toinhibit the proteasomal chymotrypsin-like activity in vitro (FIG. 8),these compounds were investigated for their potency in inhibiting intacttumor cellular proteasome activity. To do so, human prostate cancerLNCaP cells were treated with one of these compounds for the indicatedhours, followed by measuring levels of proteasome target proteins, p27,IκB-a, and Bax, and polyubiquitinated proteins.

Treatment of LNCaP cells with 50 μM of GTP-1 increased levels of p27,IκB-a, and Bax proteins at as early as 3 hours, which were significantlyincreased at 12 hours (FIG. 10A). Inhibition of proteasome activity byGTP-1 should also increase the levels of polyubiquitinated proteins,because most of the proteasome-mediated protein degradation pathwaysrequire ubiquitination (Adams, J. et al. Cancer Res, 1999, 59:2615-2622;Dou, Q. P. and Li, B Drug Resist Updat, 1999, 2:215-223; Almond, J. B.and Cohen, G. M. Leukemia, 2002, 16:433-443; Kisselev, A. F. andGoldberg, A. L. Chem Biol, 2001, 8:739-758). Indeed, GTP-1 treatmentalso caused accumulation of polyubiquitinated proteins, with the highestlevels at 12 hours (FIG. 10A). As a control, expression of actin proteinwas relatively unchanged during the treatment (FIG. 10A).

GTP-3 was more potent than GTP-1 when tested in LNCaP cells (FIG. 10A).Treatment with GTP-3 for 3 hours greatly increased levels of p27, IκB-a,and ubiquitinated proteins, which remained high or further increased for12 hours (FIG. 10A). GTP-3 transiently increased levels of Bax proteinwith the highest value at 3 hours (FIG. 10A). These results support theconclusion that amide compounds are stable and potent proteasomeinhibitors in vivo.

In another kinetic experiment, treatment of LNCaP cells with 50 μM ofGTP-4 or GTP-5 resulted in a significant increase in levels of p27 andIκB-a, with potency similar to that of (−)-EGCG (FIG. 10B). In thisexperiment, levels of Bax protein were increased only at 12 hours byGTP-4, GTP-5 or (−)-EGCG (FIG. 10B). This result demonstrates that bothGTP-4 and GTP-5 are potent proteasome inhibitors in intact tumor cells.

Previous studies have shown that overexpression of p27 (Pagano, M. etal. Science, 1995, 269:682-685), IκB-a (Verma, I. M. et al. Genes Dev,1995, 9:2723-2735), and Bax (Li, B. and Dou, Q. P. Proc Natl Acad SciUSA, 2000, 97:3850-3855) results in G₁ arrest or apoptosis. If increasedlevels of these proteasome target proteins by the synthetic EGCG analogsare functional, growth inhibition of tumor cells would be expected. Totest this possibility, GTP-4 and GTP-5 were selected, which showedsignificant potency both in vitro and in vivo (FIGS. 8, 10A, and 10B),to treat human Jurkat T cells for 24 hours, followed by flow cytometryanalysis. The G₁ population was indeed increased by 26% and 9% bytreatment with GTP-4 and GTP-5, respectively (FIG. 11). Both compoundsalso increased the sub-G1 DNA cell population by ˜15% (FIG. 11),indicating the occurrence of DNA fragmentation and cell death.

Example 5 Synthetic EGCG Analogs Inhibit Colony Formation of ProstateCancer Cells

The in vivo effects of several GTP analogs were investigated with a softagar assay that measures the transforming activity of human tumor cells.LNCaP cells were plated in soft agar (see Materials and Methods) alongwith 50 μM of GTP-1, -2, -3, (−)-EGCG (as a positive control; Smith, D.M. et al. Mol Med, 2002, 8:382-392, 2002), or the solvent (as a negativecontrol), followed by a 14 day-incubation to allow for colony formation(FIGS. 12A and 12B). The solvent-treated plates allowed for thedevelopment of ˜500 colonies, while treatment with GTP-1, -2 or -3completely blocked LNCaP transformation capability, with potency similarto that of natural (−)-EGCG (FIGS. 12A and 12B). These data suggest thatalthough GTP-1, -2 and -3 are less potent in vitro than (−)-EGCG (FIG.8), these analogs are potent in vivo.

In a second experiment, LNCaP cells were plated in soft agar along with10 μM of each indicated GTP or solvent (H₂O), followed by a 14day-incubation to allow for colony formation (FIG. 21A). Thesolvent-treated plates allowed for the development of −4,400 colonies,while synthetic (+)-EGCG treatment abolished formation of nearly all thecolonies (99.5% inhibition), similar to the natural (−)-EGCG (99.0%inhibition; FIGS. 21A and 21B). As expected, the inactive syntheticBn-(+)-EGCG, which cannot inhibit the proteasome activity (FIGS. 16A,17A, 17B, and 17C), had little inhibitory activity on colony formation(˜10% inhibition). The synthetic (+)-GCG compound was found to inhibit91.5% of colony formations, however, it was not as potent as thesynthetic (+)-EGCG or the natural (−)-GCG (99% inhibition; FIGS. 21A and21B). Colonies were quantified with an automated counter and presentedas mean values from triplicate independent experiments. Error barsdenote standard deviations. These data demonstrate that synthetic GTPs,especially (+)-EGCG, can inhibit prostate cancer cell growth and colonyformation in a semi-in situ assay.

Example 6 Synthetic EGCG Analogs Induce Accumulation of ProteasomeTarget Proteins Preferentially in Tumor and Transformed Over Normal andNon-Transformed Cells

Immortalized, non-transformed normal natural killer (NK) cells (YT line)(Drexler, H. G. et al. Leuk Res, 2000, 24:881-911) and human leukemicJurkat T cells (as a control) were treated with either (−)-EGCG (as acomparison) or (−)-EGCG-amide for up to 12 hour. Levels of IκB-α proteinwere significantly increased in Jurkat T cells by either (−)-EGCG or itsamide (up to 6- and 8-fold, respectively, at 6 hours; FIGS. 13A and13B). In contrast, IκB-α expression in non-transformed NK cells wasunchanged during each treatment, although the basal level (and themobility) of IκB-α protein was higher in NK cells (FIGS. 13A and 13B).Levels of p27 in Jurkat cells were increased by 17-fold after (−)-EGCGtreatment; no p27 expression was detected in NK cells, even aftertreatment (FIG. 13A). These data support the conclusion that EGCGanalogs can inhibit the proteasome activity selectively in tumor overnon-transformed cells (Nam, S. et al. J Biol Chem, 2001,276:13322-13330).

When the selectivity of the commercial tripeptidyl proteasome inhibitorLLL was investigated, increased levels of ubiquitinated proteins withsimilar kinetics in both Jurkat and NK cells were found (FIG. 13C),suggesting non-tumor selectivity. Therefore, EGCG analogs have greaterselectivity than LLL in leukemic Jurkat T over non-transformed NK cells.

Normal WI-38 and SV40-transformed VA-13 cell lines were treated with 25μM of (−)-EGCG, (−)-EGCG-amide, (+)-EGCG, (+)-EGCG-amide, or GTP-1 foreither 12 hours or 36 hours. It was found that at 12 hours (−)-EGCG,(−)-EGCG-amide, (+)-EGCG, (+)-EGCG-amide, and GTP-1 significantlyincreased levels of p27 protein, while at 36 hours (−)-EGCG accumulatedp27 levels (FIGS. 14A and 14B). In contrast, p27 levels in normal WI-38cells were slightly increased only by (−)-EGCG-amide, but not any othertested compounds at 36 hours (FIGS. 14A and 14B). These data suggestthat the tested EGCG analogs may have the ability to inhibit theproteasome activity preferentially in tumor and transformed cells versusnormal or non-transformed cells.

Example 7 Synthesis of Green Tea Polyphenols (GTPs) A. EnantioselectiveSynthesis of GTPs

¹H and ¹³C NMR spectra were recorded on Varian 400 or 300 MHzspectrometers. Spectra were referenced to residual chloroform (δ 7.26ppm, ¹H; δ 77.0 ppm, ¹³C), acetone (δ 2.04 ppm, ¹H, 29.8 ppm, ¹³C) ortetramethylsilane (δ 0.00 ppm, ¹H and ¹³C). Chemical shifts are reportedin ppm (δ); multiplicities are indicated by s (singlet), d (doublet), t(triplet), q (quartet), m (multiplet) and br (broad). Couplingconstants, J are reported in Hertz. [a]_(D) were measured on a JASCODIP-140 Polarimeter. Chemicals were used as obtained from commercialsources unless specified otherwise. THF was freshly distilled overNa/benzophenone and used immediately. CH₂Cl₂ was freshly distilled overCaH₂ and used immediately.trans-5,7-bis-benzyloxy-2-[3,4,5-tris(benzyloxy)phenyl]-chroman-3-ol,and tris(benzyloxy)benzoic acid chloride were prepared by literaturemethods. (+)-EGCG and the fully benzyl protected (+)-EGCG [Bn-(+)-EGCG]were prepared according to previously reported procedures.

B. Preparation of (+)-GCG

(+)-GCG was prepared according to Smith et al. (Smith, D. M. et al.Molecular Medicine, 2002, 8(7):382-392). To a solution of(+)-[2R,3S]-5,7-bis(benzyloxy)-2-[3,4,5-tris(benzyloxy)phenyl]chroman-3-ol(145 mg, 190 μmol, 1.0 equiv) in CH₂Cl₂ (15 mL) was addeddimethylaminopyridine (58 mg, 470 μM, 2.5 equiv) under N₂. The solutionwas cooled to 0° C., and 3,4,5-tris(benzyloxy)benzoic acid chloride (131mg, 285 μmol, 1.5 equiv) was added. The mixture was allowed to warm toroom temperature and stirred for 16 hours. Saturated NaHCO₃ aqueoussolution (20 mL) was added and the mixture was stirred at roomtemperature for an additional 1 hours. The layers were separated, andthe aqueous layer was extracted with EtOAc (4×25 mL). The combinedorganic phase was washed with brine (20 mL), dried over Na₂SO₄, and wasconcentrated by rotary evaporator and vacuum drying to give crudeproduct which was purified by silica gel chromatography(C₆H₆/EtOAc=100/1) to afford pure product 195 mg (87%) of the fullybenzyl protected gallate ester as a white solid. [a]_(D)=22.17 (c1.09,CHCl₃); NMR (300 MHz, CDCl₃): 7.31 (m, 42H), 6.70 (s, 2H), 6.32 (s, 1H),6.31 (s, 1H), 5.48 (m, 1H), 5.12 (m, 1H), 5.01 (m, 16H), 2.99 (dd,J=16.8, 5.2, 1H), 2.84 (dd, J=16.8, 10.2, 1H); ¹³C NMR (75 MHz, CDCl₃):165.07, 158.92, 157.64, 154.78, 152.86, 152.39, 142.54, 138.36, 137.66,137.33, 136.76, 136.71, 136.47, 133.38, 128.59, 128.53, 128.48, 128.43,128.39, 128.25, 128.15, 128.08, 128.02, 127.98, 127.94, 127.90, 127.81,127.75, 127.62, 127.50, 127.48, 127.21, 124.92, 109.06, 106.23, 101.29,94.26, 93.78, 78.41, 75.10, 75.05, 71.22, 71.13, 70.09, 69.90, 69.80,24.00.

To a solution of the fully benzyl protected gallate ester (100 mg, 84.8μmol) obtained above in MeOH/THF (10/10 mL) was added Pd(OH)₂ (105 mg,20% on carbon). The mixture was stirred at room temperature under H₂ andmonitored by TLC. When the starting material was consumed (in about 4hours), the mixture was filtered through cotton to remove the catalyst,and eluted with acetone (5.0 mL). The combined eluate was concentratedby rotary evaporator and vacuum drying to give the crude product whichwas purified by silica gel chromatography (EtOAc/CH₂Cl₂=2.5/1) to affordthe pure (+)-GCG, 28.6 mg (74%) as a white solid. [a]_(D)=11.78 (c 0.78,THF); NMR (300 MHz, acetone-d/D₂O=2/1): 6.94 (s, 2H), 6.44 (s, 2H), 5.98(d, J=2.5, 1H), 5.88 (d, J=2.5, 1H), 5.23 (q, J=6.6, 1H), 4.93 (d,J=6.6, 1H), 3.00 (dd, J=16.5, 5.2, 1H), 2.61 (dd, J=16.5, 6.6, 1H); ¹³CNMR (75 MHz, CDCl₃): 166.25, 156.73, 156.33, 155.24, 145.66, 145.22,138.69, 132.97, 129.64, 120.34, 109.47, 106.07, 98.70, 95.87, 94.81,78.30, 70.27, 24.25. The compound had identical NMR spectra as thecommercially available natural (−)-GCG (Sigma, [a]_(D)=−12.44 (c 0.8,THF).

C. Synthesis ofcis-(±)-5,7-Bis-benzyloxy-2-(3,4,5-tris-benzyloxyphenyl)-chroman-3-yl-amine((±)-2) and the enantiomers (+)-2 and (−)-2

To a solution of(±)-trans-5,7-bis-benzyloxy-2-[3,4,5-tris(benzyloxy)phenyl]chroman-3-ol((±)-1) (152.6 mg, 0.2 mmol) in THF (8.0 mL) was addedtriphenylphosphine (262.3 mg, 1.0 mmol), diethylazodicarboxylate (174.2mg, 1.0 mmol) and diphenylphosphoryl azide (269.7 mg, 0.98 mmol) at roomtemperature. The solution was stirred at room temperature for 2 hours.EtOAc (20 mL) and H₂O (10 mL) were added and stirred for an additional10 minutes. The layers were separated and the aqueous layer wasextracted with EtOAc (4×50 mL). The combined organic phase was washedwith brine (15 mL) and dried over anhydrous Na₂SO₄. The solution wasconcentrated by rotary evaporator and vacuum drying to give the crudeproduct which was purified by silica gel chromatography(C₆H₆/EtOAc=100/1) to afford the mixture of desired product and theelimination by-product (ratio is about 1/1). This mixture was dissolvedin THF (8.0 mL), after which triphenylphosphine (85 mg) and H₂O (65 mg)were added. The solution was refluxed overnight followed by addition ofH₂O (180 mg) and then refluxed for 3 hours. The solution wasconcentrated by rotary evaporator and vacuum drying to give the residuewhich was purified by silica gel chromatography (C₆H₆/EtOAc=5/1) toafford pure product 42 mg (28%) of (±)-2 as a white solid. ¹H NMR: (300MHz, CDCl₃), 7.36 (m, 25H), 6.74 (s, 2H), 6.29 (s, 2H), 5.16 (s, 4H),5.09 (s, 2H), 5.04 (s, 4H), 4.95 (s, 1H), 3.34 (m, 1H), 2.99 (dd,J=16.8, 5.2, 1H), 2.86 (dd, J=16.8, 1.9, 1H), 1.06 (s, br, 2H). ¹³C NMR:(75.5 MHz, CDCl₃), 158.47, 158.17, 154.86, 152.61, 137.79, 137.65,136.85, 136.83, 136.74, 134.47, 128.46, 128.44, 128.37, 128.34, 128.19,128.02, 127.86, 127.76, 127.72, 127.68, 127.41, 127.39, 127.04, 105.72,101.44, 94.50, 93.83, 78.93, 75.16, 71.24, 70.08, 69.86, 48.27, 28.32.By employing the same procedure as described above, and starting fromthe appropriate enantiomer of 1,(−)-[2R,3]-5,7-bis-benzyloxy-2-(3,4,5-tris-benzyloxyphenyl)chroman-3-yl-amine((−)-2), [α]_(D)=−14.6 (c=3.0, CHCl₃) and(+)-[2S,3S]-5,7-bis-benzyloxy-2-(3,4,5-tris-benzyloxyphenyl)chroman-3-yl-amine((+)-2), [α]_(D)=16.8 (c=3.5, CHCl₃) were prepared with identical NMRspectra as (±)-2.

D. Synthesis ofcis-(±)-3,4,5-Tris-benzyloxy-N-[5,7-bis-benzyloxy-2-(3,4,5-tris-benzyloxyphenyl)-chroman-3-yl]-benzamide((±)-4) and the enantiomers (+)-4 and (−)-4

To a solution of (±)-2 (50 mg, 65.6 mmol) in CH₂Cl₂ (6.0 mL) was addedDMAP (20 mg, 164 mmol). The solution was cooled to 0° C., andtris(benzyloxy)benzoic acid chloride (45 mg, 98.4 mmol) was added. Themixture was stirred at 0° C. for 2 hours, allowed to warm to roomtemperature, and was stirred overnight. Saturated NaHCO₃ aqueoussolution (10 mL) was then added and stirred for 1 hour, layers wereseparated, and the aqueous layer was extracted with EtOAc (5×20 mL). Thecombined organic phase was washed with brine (15 mL) and dried overanhydrous Na₂SO₄. The solution was concentrated by rotary evaporator andvacuum drying to give the crude product which was purified by silica gelchromatography (C₆H₆/EtOAc=50/1) to afford the pure product 62.4 mg(81%) of (±)-4 as a white solid. ¹H NMR: (400 MHz, CDCl₃), 7.33 (m,40H), 6.87 (s, 2H), 6.80 (s, 2H), 6.36 (m, 2H), 6.31 (d, J=8.8, 1H),5.06 (m, 18H), 3.11 (dd, J=17.3, 5.2, 1H), 3.03 (dd, J=17.3, 2.8, 1H).¹³C NMR: (100.6 MHz, CDCl₃), 166.54, 158.63, 158.01, 154.90, 152.78,152.42, 141.13, 138.11, 137.48, 137.17, 136.63, 136.52, 136.49, 136.29,133.20, 129.74, 128.50, 128.42, 128.32, 128.29, 128.01, 127.96, 127.82,127.76, 127.72, 127.62, 127.49, 127.39, 127.00, 106.76, 105.82, 101.75,94.73, 94.27, 77.49, 75.15, 75.09, 71.42, 71.34, 70.18, 69.93, 45.99,26.82. By employing the same procedure as described above, and startingfrom the appropriate enantiomer of 2,(−)-[2R,3R]-3,4,5-tris-benzyloxy-N-[5,7-bis-benzyloxy-2-(3,4,5-tris-benzyloxyphenyl)-chroman-3-yl]-benzamide((−)-4), [α]_(D)=−14.5 (c=4.7, CHCl₃) and(+)-[2S,3S]-3,4,5-tris-benzyloxy-N-[5,7-bis-benzyloxy-2-(3,4,5-tris-benzyloxyphenyl)-chroman-3-yl]-benzamide((+)-4), [α]_(D)=10.9 (c=4.5, CHCl₃) were prepared with identical NMRspectra as the (±)-4.

E. Synthesis ofcis-(±)—N-[5,7-Dihydroxy-2-(3,4,5-trihydroxyphenyl)-chroman-3-yl]-3,4,5-trihydroxybenzamide((±)-5 and the enantiomers (+)-5 and (−)-5

To a solution of (±)-4 (45.5 mg, 38.6 μmol) in THF/MeOH (4.5/4.5 mL) wasadded Pd(OH)₂ [20 wt. % Pd (dry basis) on carbon] (50 mg). The mixturewas stirred at room temperature under H₂ and monitored by TLC. Two hourslater, the starting material was consumed according to TLC analysis. ThePd catalyst was removed by filtering through cotton, and eluted withacetone (4.0 mL). The combined elute was concentrated by rotaryevaporator and vacuum drying to give the crude product which waspurified by reverse phase column chromatography (MeOH/H₂O=4/6) to affordthe pure product 14.5 mg (82%) of (±)-5 as a pale yellow solid. ¹H NMR:(400 MHz, acetone-d/D₂O=2/1), 7.00 (d, J=8.0, 1H), 6.68 (s, 2H), 6.54(s, 2H), 5.98 (d, J=2.2, 1H), 5.92 (d, J=2.2, 1H), 5.01 (d, J=1.4, 1H),4.53 (m, 1H), 2.87 (dd, J=16.8, 5.2, 1H), 2.75 (dd, J=16.8, 3.0, 1H).¹³C NMR: (100.6 MHz, acetone-d/D₂O=2/1), 168.13, 156.58, 156.42, 155.60,145.50, 145.12, 136.68, 132.32, 129.99, 124.82, 107.03, 105.43, 99.29,96.18, 95.27, 77.26, 47.57, 26.32. By employing the same procedure asdescribed above, and starting from the appropriate enantiomer of 4,(−)-[2R,3R]-N-[5,7-Dihydroxy-2-(3,4,5-trihydroxyphenyl)-chroman-3-yl]-3,4,5-trihydroxybenzamide((−)-5), [α]_(D)=−112 (c=0.95, acetone/H₂O=2/1) and (+)-[2S,3S]-N-[5,7-Dihydroxy-2-(3,4,5-trihydroxyphenyl)-chroman-3-yl]-3,4,5-trihydroxybenzamide((+)-5), [α]_(D)=101 (c=0.67, acetone/H₂O=2/1) were prepared withidentical NMR spectra as the (±)-5.

Example 8 (−)-EGCG Kinetics

A kinetics experiment showing the decrease in chymotrypsin-like activityover time was conducted. (−)-EGCG at 1 μM potently inhibited thechymotrypsin-like activity of a purified eukaryotic (rabbit) 20Sproteasome in a time-dependent manner: 35% at 5 min, 62% at 30 min, and70-80% after 1 to 3 hours (FIG. 7), which is characteristic for amechanism-based inhibitor. This result further demonstrates that themode of (−)-EGCG action is irreversible inhibition.

(−)-EGCG at 1 μM was incubated with eukaryotic 20S proteasome (0.02 μg)and suc-LLVY-AMC (20 μM) for the times indicated in FIG. 7. Thechymotrypsin activity was measured and graphed in FIG. 7. Values aremeans from 4 independent experiments, and error bars represent standarddeviations.

Example 9 Proteasome Inhibition by EGCG and its Analogs

There are two aspects of proteasome inhibition by (−)-EGCG. First, itwas demonstrated that (−)-EGCG irreversibly inhibits thechymotrypsin-like activity of the proteasome in a time-dependant manner(FIGS. 6 and 7), so it is plausible that a nucleophilic attack of theester bond carbon of (−)-EGCG occurs. Secondly, in order for the aboveevent to occur, (−)-EGCG must bind to the active site in the appropriateorientation and conformation that allows for attack of the carbonylcarbon of (−)-EGCG to take place (FIG. 2A).

(−)-EGCG can bind the proteasome's chymotrypsin active site in anorientation and conformation that is well suited for nucleophilic attackas described by the following model. First, eight H bonds can formbetween (−)-EGCG and the β5 subunit (FIGS. 2D and 2E). Second, favorablehydrophobic surface interactions exist (tyrosine-like mimic in S1pocket) (FIGS. 2D and 2E). Third, there is a large potential van derWalls contact surface area (FIG. 2C). Fourth, the calculated free energyvalues are favorable for binding of (−)-EGCG to the proteasome (FIG.3A). Fifth, it is likely that the scissile bond of (−)-EGCG is strained,suggesting lowering of the activation energy for the formation of thetetrahedral intermediate in the proposed acylation reaction (FIG. 4).Finally, it was observed that one of the two docked structures of lowestfree energy for (−)-EGCG had its electrophilic carbonyl carbon 3.18 Åfrom the hydroxyl group of Thr 1 (FIGS. 2A and 2B). All these propertiesdemonstrated by this reported docking model have supplied an attractive,empirically directed, analog supported model of proteasome inhibition bythe green tea polyphenol (−)-EGCG.

Novel analogs of naturally occurring tea polyphenols have beensynthesized. Such analogs have been tested for their inhibitorypotencies against the proteasome. Two analogs of (−)-EGCG weresynthesized. The first analog, (+)-EGCG, was the enantiomer (mirrorimage) of the natural (−)-EGCG, having the 2R, 3R configuration insteadof the natural 2S, 3S configuration. [FIG. 15, (−)-EGCG vs. (+)-EGCG].The second analog, Bn-(+)-EGCG, was synthesized with all eight hydroxylsprotected by benzyl groups. This should eliminate any hydrogen bondingcreated by the hydroxyls in (+)-EGCG while maintaining the integrity ofthe ester bond. In addition the enantiomer of the natural (−)-GCG wasalso synthesized, giving (+)-GCG with the 2R, 3S configuration [FIG. 15,(−)-GCG and (+)-GCG].

The ability of each purified synthetic GTP analog to inhibit thechymotrypsin-like activity of purified 20S proteasome, using the naturalGTPs for comparison was determined. In this experiment, the synthetic(+)-EGCG at 1 μM inhibited 86% of the proteasomal activity, similar tothat of its natural counterpart, (−)-EGCG (FIG. 16A). However, thebenzyl-protected compound, Bn-(+)-EGCG, did not significantly inhibitthe proteasome activity even at 10 μM concentrations (FIG. 16A),demonstrating the requirement of one or more hydroxyl groups for theproteasome-inhibitory activity of EGCG. The synthetic (+)-GCG was foundto inhibit 70% of the proteasomal activity at 1 μM, similar to thenatural (−)-GCG (FIG. 16A). Both forms of EGCG are more potent than thetwo GCG analogs (FIG. 16A). As a control in this experiment, awell-known tripeptidyl proteasome inhibitor LLnL at 1 and 10 μMinhibited 20 and 70% proteasomal activity, respectively (FIG. 16A).

To further determine the proteasome-inhibitory activities of thesynthetic GTP analogs, multiple concentrations of each GTP was used inorder to measure their one-half maximal inhibition values (IC₅₀s). AnIC₅₀ of 210 nM was found for the synthetic (+)-EGCG, similar to thecontrol (−)-EGCG (FIG. 2B). In addition, the (+)-GCG compound, similarto the natural (−)-GCG, produced an IC₅₀ value of 410 nM, nearly twicethat of (+)-EGCG (FIGS. 2B and 2C). Dose-dependant inhibition ofpurified 20S proteasome by (+)-EGCG and (−)-EGCG (B), or (+)-GCG and(−)-GCG (C) is graphed on a log plot. All values are means ofindependent triplicate experiments. Error bars denote standarddeviations (error bars were not included on B and C for clarity ofpresentation)

To determine the specificity of the synthetic GTP analogs, their effectswere tested on the chymotrypsin-like and trypsin-like activities of the26S proteasome in a Jurkat cell lysate. Similar to inhibition of thepurified 20S proteasome, the synthetic (+)-EGCG at 10 μM inhibited 76%of the chymotrypsin-like activity of 26S proteasome in cell lysates(FIG. 17A). In contrast, the Bn-(+)-EGCG analog could not inhibit theproteasomal chymotrypsin activity at all (FIG. 3A). (+)-GCG at the sameconcentration inhibited ˜50% of the chymotrypsin-like activity. Thepotencies of (+)-EGCG and (+)-GCG were similar to those of their naturalpartners, respectively (FIG. 17A). However, none of the synthetic ornatural GTPs could significantly inhibit the proteasomal trypsin-likeactivity in the Jurkat cell extract (FIG. 3B). Furthermore, none of thesynthetic or control natural compounds could inhibit more than 15% ofactivity of a purified calpain enzyme (FIG. 17C). These data stronglysuggest that the synthetic GTPs with ester bonds selectively inhibit thechymotrypsin-like activity of the proteasome. Values are meantriplicates and error bars denote standard deviations.

A. Accumulation of Proteasome Target Proteins P27 and IκB-a andInduction of Tumor Cell G₁ Arrest by Synthetic GTPs

Having discovered that the synthetic GTP analogs are potent and specificinhibitors of the proteasomal chymotrypsin-like activity in vitro (FIGS.16A, 17A, 17B, and 17C), it was then determined whether the syntheticcompounds could also be effective in inhibiting intact tumor cellproteasome activity. Because inhibition of the proteasome activity intumor cells would result in increased levels of proteasome targetproteins, levels of p27 and IκB-a proteins were measured by Western blotassay in Jurkat T cells treated with each GTP. After a 12 hourtreatment, (+)-EGCG at 10 μM accumulated p27 and IκB-a levels by 3.5-and 3.1-fold, respectively (FIG. 18A). In comparison, the natural(−)-EGCG accumulated p27 and IκB-a, to a slightly lesser extent, by 2.7-and 2.1-fold, respectively (FIG. 18A). In addition, synthetic (+)-GCGincreased p27 and IκB-a levels by 3.7- and 3.3-fold, respectively,similar to the results from (−)-GCG treatment (FIG. 18A).

The above experiment was also performed in prostate cancer LNCaP cells.The synthetic (+)-EGCG increased the levels of p27 and IκB-a by 3.1 and2.6-fold, respectively, comparable to the effects of (−)-EGCG (FIG.18B). A 5.8- and 3.7-fold increase in p27 and IκB-α. levels,respectively, was observed after treatment with (+)-GCG (FIG. 18B). Thecontrol compound (−)-GCG did not accumulate as much p27 protein as thesynthetic compound did, although both compounds accumulated similarlevels of IκB-a (FIG. 18B).

Molecular masses of IκB-a and actin are 40 and 43 kDa, respectively.Relative Density (RD) values are normalized ratios of the intensities ofp27 and IkB-a band to the corresponding actin band. Data isrepresentative of at least three independent experiments

It has been shown that overexpression of p27 and IκB-a results in G₁arrest. If increased levels of these proteasome target proteins bysynthetic GTPs are functional, it would be expected to see growth arrestof tumor cells in G₁ phase. To test this hypothesis, asynchronousprostate LNCaP cells were treated with 10 μM of synthetic (+)-EGCG or(+)-GCG, along with each respective natural GTP as control, for 24hours. Cells were then harvested, and analyzed by cell cycle analysis byflow cytometry. The amount of G₁, S and G₂/M cell populations wasdemonstrated by DNA histogram. Growth arrest was determined by theincrease in the percentage of the G₁ population.

It was observed that treatment with each of the GTPs significantlyincrease the G₁ population, accompanied by a reduction in S and G₂/Mphase cell populations. Specifically, the synthetic (+)-EGCG increasedthe G₁ population by 18%, while (−)-EGCG, (+)-GCG and (−)-GCG allinduced ˜14% G₁ arrest (FIG. 19, C for control). Therefore, thesynthetic GTPs have the ability to inhibit prostate tumor cell growth,with potency either similar to, or even greater than, that of thenatural compounds. Data is representative of at least three independentexperiments.

B. Accumulation of the Pro-Apoptotic Bax Protein and Activation of TumorCell Apoptotic Program by Synthetic GTPs

The pro-apoptotic protein Bax is another target protein of theproteasome. To investigate whether synthetic tea polyphenols have theability to induce Bax-associated cancer cell apoptosis, two humanprostate cancer cell lines, LNCaP and DU145, were used. LNCaP cellsexpress much higher levels of Bax protein than DU145 cells. Thecomparison between these two cell lines provides an excellent model forstudying the role of Bax in the process of GTP-mediated proteasomeinhibition and apoptosis induction.

Exponentially growing LNCaP and DU-145 prostate cells were harvested andthe levels of Bax protein was detected by Western blot analysis (FIG.20A). A high-molecular-weight band, recognized by the anti-Bax antibody,is indicated by an arrowhead. Although its nature remains unknown, thishigh-molecular-weight band can be used as a loading control. LNCaP orDU-145 cells were treated for 24 hours with either H₂O(C, for control)or 10 μM (−)-EGCG or (+)-EGCG (FIG. 20B). Cells were then harvested andWestern-blotted with specific antibodies to Bax, actin (FIG. 20B) orPARD (FIG. 20D). Data are representative of at least three independentexperiments.

High Bax protein (21 kDa) levels in LNCaP cells, and very low Baxexpression in DU145 cells were also observed (FIG. 20A). Treatment ofLNCaP cells for 24 hours with synthetic (+)-EGCG at 10 μM significantlyincreased Bax protein levels, similar to the effect of the natural(−)-EGCG (FIG. 20B). In contrast, neither (+)-EGCG nor (−)-EGCG was ableto increase the levels of Bax protein in DU145 cells (FIG. 20B).

To examine whether (+)-EGCG could induce apoptosis only in LNCaP, butnot DU-145, cells, both cell lines were treated with 10 μM (+)-EGCG for0, 24, 48 or 72 hours, followed by performance of cell-free caspase-3activity assay. Cells were harvested at each sequential time-point andactivity was determined by incubating whole cell extracts with caspase-3substrate and measuring free AMCs (FIG. 20C). All the time-points werenormalized to 0 hours. (+)-EGCG activated caspase-3 in LNCaP cells in atime-dependent manner: by 2.5-, 3.5- and 6-fold at 24, 48 and 72 hours,respectively (FIG. 20C). In contrast, little caspase-3 activity wasdetected in (+)-EGCG-treated in DU145 cells: only-2-fold induction at 72hours (FIG. 20C). In the same experiment, (−)-EGCG at 10 μM was used asa control. It was found that the potency of (+)-EGCG to activatecaspase-3 was comparable to that of (−)-EGCG in both LNCaP and DU145cells (FIG. 20C). Consistent with that finding, (+)-EGCG, as well as(−)-EGCG, induced the apoptosis-specific PARD cleavage only in LNCaP,but not DU145 cells (FIG. 20D). Therefore, the abilities of (+)-EGCG toactivate apoptotic program in these prostate cancer cell lines arecorrelated well to their abilities to accumulate Bax protein levels to acritical high threshold (compare FIGS. 20C and 20D vs. 20B). Values aremeans of triplicate independent experiments and error bars denotestandard deviations. These data suggest that EGCG accumulated Baxprotein plays an essential role in activating caspases and inducingapoptosis in human prostate cancer cells.

The physiological concentration of a single GTP (such as EGCG) has beenobserved to be in the single digit μM to high nanomolar ranges.Therefore, if a GTP of interest is to function physiologically, themechanism of action must therefore occur at concentrations similar tothose found for GTPs in serum.

Synthesis of GTP analogs should also prove beneficial for determiningwhich mechanisms are responsible for the observed anti-cancer effects.For example, it is possible that the kinase-inhibitory activity of EGCGcould be removed in some EGCG analogs while retaining theproteasome-inhibitory activities, and vice-versa. These synthetic GTPanalogs could therefore help to solve the problem of distinguishing theimportant mechanistic properties of tea polyphenols.

Synthetic GTPs are active and comparable to their natural counterpartsin biological assays. The proteasome-inhibitory potency of the synthetic(+)-EGCG was found to be similar to, and sometimes higher than, that ofthe natural stereoisomer (−)-EGCG (FIGS. 2-7). A similar characteristicwas also found for the (+)-GCG compound and its control (−)-GCG.However, the benzyl-protected compound, Bn-(+)-EGCG, which has noavailable hydroxyls for hydrogen binding, can not inhibit the proteasomedespite the presence of the ester-bond that is susceptible to anucleophilic attack by the proteasome's N-terminal threonine. Thissuggests that hydrogen bonding to some or all of EGCG's eight OH groupsare important in binding to the proteasome active site for inhibition.However, introduction of eight such benzyl groups would also no doubtadd a lot of mass to EGCG as well as cause other steric difficultiesduring proteasomal binding in addition to the elimination of hydrogenbonding potentials.

The aforementioned experiments demonstrate that the syntheticenantiomers of these two GTPs, (+)-EGCG and (+)-GCG, at least did notloose any, and even may have gained some potency in regards toproteasome inhibition. FIGS. 17A, 17B, and 17C also demonstrate that thespecificity profile is also the same in regards to three differentprotease activities: chymotrypsin-like, trypsin-like, and calpain. Thisat least suggests that the synthetic stereoisomers are inhibiting via asimilar mechanism as the natural occurring compounds. Normally, mostbiological processes show chiral discrimination, where enantiomersdisplay different biological activities. While it is too early tospeculate on the reason for the lack of chiral discrimination in thepresent case, an argument can be made that the presence of the twotrihydroxyphenyl rings in EGCG (or GCG) may have rendered the moleculepseudo-symmetric in terms of its binding to the active site ofproteasome.

It has been suggested that under in vivo conditions, GTPs, specifically(−)-EGCG, are unstable and can be degraded or altered, quickly makingthem unavailable for inhibition of enzymatic activities. It was thenthought that synthetic GTPs might have identical potencies as naturalones, but with increased stability. To test this idea synthetic andnatural GTP compounds were used in both suspension (Jurkat) and solidtumor (LNCaP) cell lines to determine their ability to accumulateproteins that are degraded by the proteasome. In Jurkat cells thishypothesis seemed at least partially fulfilled because there was nearlya 30% increase in p27 levels and a 47% increase in IκB-a levels in cellstreated with (+)-EGCG, as compared to (−)-EGCG (FIG. 18A). However, theaccumulation of p27 and IκB-a levels in LNCaP cells was nearly identicalfor both (+)-EGCG as well as (−)-EGCG (FIG. 18B). The (+)-GCG compoundalso increased the levels of both proteins to higher levels in both celllines as compared to the (−)-GCG compound, except p27 in Jurkat cells(FIGS. 18A and 18B). Though these differences are marginal it at leastsuggests the possibility that synthetic enantiomers may have bettereffects in animal models and clinical trials than do their naturalcounterparts, possibly due to an increase in drug stability in vivo.

In agreement with p27 and IκB-a accumulation in the in vivo experiments,cell cycle arrest was also observed after LNCaP cells were treated withthese GTPs. Again, (+)-EGCG was found to be the most potent compound andinduced an 18% G₁ arrest, compared to the positive control (−)-EGCGwhich induced 14% G₁ arrest, an approximately 30% increase just bychanging the stereochemistry of the natural compound (FIG. 19). Again,this could suggest an increased stability of the synthetic compoundmight be responsible for increased ability to induce growth arrest.

To better determine whether these synthetic compounds induceBax-dependent apoptosis, a pair of prostate cancer cell lines witheither high or low Bax protein expression were used. It has beendemonstrated that proteasome inhibitors can induce tumor cell death viaaccumulation of the pro-apoptotic Bcl-2 family member Bax. GTPs havebeen shown to induce apoptosis, so it was hypothesized that thesynthetic EGCG analog should be able to accumulate levels of Bax andinduce apoptosis in LNCaP cells which contain high basal levels of Bax,but should not induce apoptosis in DU-145 cells which express low Baxprotein. Indeed, when these two prostate cancer cell lines were treatedwith either (+)-EGCG or natural (−)-EGCG, apoptosis was induced by bothcompounds in LNCaP cells but not in DU-145 cells, as judged by caspase-3activation and PARP cleavage (FIG. 20A). This suggests that thesynthetic EGCG analog can induce apoptosis in a Bax-dependant manner,supporting the conclusion that apoptosis would have been initiated viainhibition of proteasome-mediated Bax degradation.

The desired effect of any anti-tumor compound includingcancer-preventative agents is to inhibit tumor growth and formation insitu. An assay developed to semi-mimic cellular growth in tissue is thecolony forming soft agar assay. The synthetic GTPs that can inhibit theproteasome activity and cell cycle progression as well as induce celldeath should be able to inhibit colony formation in a soft agar assay.Indeed, when LNCaP tumor cells were cultured in the presence ofsynthetic GTPs, an almost complete inhibition of colony formation wasobserved, as compared to the solvent control and the inactiveBn-(+)-EGCG (FIG. 21A).

Example 10 (−)-EGCG and Analogs Docking Studies

(−)-EGCG's susceptibility to a nucleophilic attack is demonstrated inthe HPLC results that showed that the proteasome could attack anddegrade (−)-EGCG. Kinetic analysis and X-ray diffraction studies usingthe specific proteasome inhibitor lactacystin have demonstrated that theester bond of this inhibitor covalently modifies the N-terminalthreonine of the β5 subunit, which is critical for proteasomeinhibition. Since (−)-EGCG contains an ester bond (FIG. 1) and inhibitsthe proteasome irreversibly in a time-dependent manner (FIGS. 6 and 7),it is probable that a lactacystin-like reaction occurs with (−)-EGCG.

A. Automated Docking of (−)-EGCG to the β5 Subunit of 20S Proteasome

To build a model for how (−)-EGCG binds to the proteasome, which willallow for nucleophilic attack, automated docking studies were performed.Knowledge of the enzyme kinetics discussed above can help direct dockingsolutions that would allow covalent modification and inhibition of theproteasome.

Before acylation of Thr 1's hydroxyl by (−)-EGCG can occur, (−)-EGCGmust bind to the β5-active site in a conformation that would allow areaction to occur between the two atoms involved. Binding of (−)-EGCG inan appropriate orientation and conformation is therefore necessary forester bond scission because the presence of an ester bond alone isinsufficient to inhibit the proteasome. This is demonstrated by benzylprotected-EGCG, which has an ester-bond, but cannot inhibit theproteasome. In addition, several small molecular weight molecules thatcontain ester bonds, including methyl acetate, benzyl hydroxybenzoate,and methyl gallate, cannot inhibit the proteasome.

Docking modes were therefore chosen based on the following twopre-defined criteria. First, the distance between the carbonyl carbon of(−)-EGCG and the hydroxyl of Thr 1 must be between 3-4 Å. Secondly, thering system must be located within the S1 pocket. Based on these twocriteria, the lowest docked free energy (negative G) was chosen for sucha bound conformation. After docking (−)-EGCG to the β5 chymotrypsinactive site, the ester bond of (−)-EGCG in one of the two lowest freeenergy docked structures could be easily found oriented directly overthe Thr 1 side-chain and the ester bond-carbon was located 3.18 Å awayfrom the hydroxyl of Thr 1 (FIGS. 2A and 2B). Such anorientation/conformation of the inhibitor is well suited fornucleophilic attack and is structurally achievable, which satisfies thefirst pre-defined criterion.

In addition, the fairly hydrophobic AC rings of (−)-EGCG (see FIG. 1)were oriented in the S1 pocket of the β5 subunit, the B ring projectedup into solvent, bridging the two walls of the binding cleft, and thegallate (G) group sat above Ser 131 (FIGS. 2A and 3A). (−)-EGCG filledthe majority of the binding cleft which was seen by drawing a wateraccessible mesh surface around (−)-EGCG when docked into the bindingsite as depicted by a ribbon structure of the β5 subunit (FIGS. 2B and2C). The occupancy of the S1 pocket satisfied the second criterion, andthe docking mode chosen possessed a free energy of −10.52 kcal/mol. Thisresultant model supports the hypothesis that (−)-EGCG first binds to theβ5 active site and then is attacked by the N-terminal threonine,rendering the proteasome inactive by acylation.

There are eight polar hydrogens and one carbonyl-oxygen on (−)-EGCG thatare available for H-bonding (see FIG. 1). It appears that all but two ofthese sites are actively participating in H-bonding. It should be notedthat a relatively loose criterion was employed to establish the presenceof a hydrogen bond because this structure is not energy minimized norhave its hydrogen bond distances been optimized.

The carbonyl oxygen of (−)-EGCG H-bonds with the side chain hydrogen onThr 1 of the β5 subunit, with a calculated hydrogen bond distance 2.59 Å(FIG. 2D). In addition, two hydroxyls on the G ring H-bond with thebackbone nitrogen atoms of Ser 131 and Gly 47 (2.82 and 2.28 Å,respectively; FIG. 2D). Furthermore, the A ring-hydroxyl of (−)-EGCG,which is further from the C ring, appears to be H-bonding with thebackbone carbonyl of Gly 47 (2.74 Å; FIG. 2D) and the backbone nitrogenof Ala 46 (3.37 Å; FIG. 2E). The other A ring-hydroxyl H-bonds with thecarbonyl oxygen of Lys 32 (3.35 Å; FIG. 2E). Finally, two of the EGCG Bring hydroxyls, which bridge the binding cleft (see FIGS. 2A and 3A),are H-bonded to the side chain of Thr 21 and the backbone nitrogen ofAla 49 (2.51 and 2.16 Å, respectively; FIG. 2E). This analysis hasidentified eight H-bonds, which seem important for (−)-EGCG binding tothe proteasome β5 subunit. Consistent with this analysis, the fullybenzyl protected-EGCG without free OH groups, which should not formH-bonds, fails to inhibit the proteasome and could not be found dockedin an orientation/conformation that met criteria 1 and 2.

The hydrophobic interactions between (−)-EGCG and the β5 subunit wereanalyzed. The chymotrypsin-like activity of the proteasome cleavespeptides after hydrophobic residues, such as the Tyr in the modelfluorogenic substrate Suc-Leu-Leu-Val-Tyr-AMC. This Tyr would bind tothe S1 hydrophobic pocket of the β5 subunit to allow for specificchymotrypsin-like cleavage of the AMC group. It seems that the A ring of(−)-EGCG mimics the Tyr residue of the proteasome peptide substrate: thehydrophobic portion of this aromatic ring is oriented in the middle ofthe S1 pocket between the side chains of Ala 49, Ala 20, and Lys 33(with distances of 3.47, 3.36 and 4.24 Å, respectively; FIG. 2F). Thisconformation allows the hydrophilic hydroxyls of the A ring to projectout of the two sides of the S1 hydrophobic pocket and participate in Hbonding as described above. In addition, the side-walls of the S1 pocketthat interact with (−)-EGCG are created by Met 45 and Val 31 (3.64 and3.54 Å; FIG. 2F). Each of these hydrophobic or partially hydrophobicresidues are less than 4.5 Å from (−)-EGCG (see FIG. 2F), suggestingthat entropically driven hydrophobic interactions might indeed occurbetween the (−)-EGCG A-C rings and the S1 pocket. Therefore, inhibitionkinetics, along with docking studies of (−)-EGCG bound to the proteasomeβ5 subunit, suggests a mechanistic model for how (−)-EGCG inhibits theproteasomal chymotrypsin-like activity.

When (−)-EGCG binds the proteasome, a saddle shape is formed between theA-C rings extending past the ester bond and back down to the gallatemoiety (FIG. 4E). The more flexible nature of the ester bond allows thisconformation to occur so that (−)-EGCG might fit the saddle-shape formedby the bottom of the binding pocket (FIG. 4F, top/right). In fact, whenthe docked conformations of all the EGCG analogs are overlapped into oneimage, this saddle shape can be easily observed (FIG. 4F). Thissaddle-shaped conformation of EGCG possibly places additional strain onthe scissile bond further lowering the activation energy fornucleophilic attack.

B. Docking of Other Natural and Synthetic EGCG Analogs

Whether this established model of (−)-EGCG inhibition could also be usedto interpret the proteasome-inhibitory properties of other EGCG analogswas also investigated. Three natural, (−)-GCG, (−)-ECG and (−)-CG, andtwo synthetic, (+)-EGCG and (+)-GCG, polyphenols were chosen, all ofwhich contain an ester bond (FIG. 1). Similar to (−)-EGCG, all of thesefive polyphenols potently inhibited the chymotrypsin-like activity ofthe rabbit 20S proteasome, with IC₅₀ values similar to those obtainedusing prokaryotic 20S proteasome (FIG. 3).

Each of these five polyphenols was docked to the 20S proteasome β5subunit, using (−)-EGCG as a comparison (FIG. 3). The B₅ subunit isrepresented with a water accessible surface and colored by atom type(O-red, N-blue, C-gray, H-gray). For each compound, a single dockingmode with the lowest free energy was selected after applying the twopreset criteria. (+)-EGCG was found to be slightly more potent than(−)-EGCG with regard to purified 20S proteasome (IC₅₀ 170 nM vs. 205 nM;FIGS. 3A and 3B).

(+)-EGCG was oriented in the proteasome β5 subunit with a seeminglysimilar mode compared to (−)-EGCG, with the A-C rings in the S1 pocket,and the B ring in solvent, bridging the binding cleft (FIGS. 3B vs. 3A).The ester bond-carbon (and gallate group) were shifted only 0.38 Å awayfrom Thr 1, as shown in FIG. 2D, but still resided over Thr 1 in asuitable position for a nucleophilic attack. [see FIG. 3B-1, for overlapof (+)-EGCG and (−)-EGCG]. The shift of this gallate group placed thecarbonyl oxygen into the binding cavity created by Arg 19 and Thr 21(also see FIG. 2E), allowing for an increased van der Waals interactionand a slightly more favorable binding free energy (−10.82 kcal/mol vs.−10.52 kcal/mol; FIGS. 3A and 3B), explaining the increased activity ofthis compound. A closer inspection revealed that (+)-EGCG had to flipover 180 degrees (in relation to the plane of the A-C rings) in order toattain a similar orientation/conformation. It is known that (−)-EGCG and(+)-EGCG have (2R,3R) and (2S,3S) stereochemistry, respectively (FIG.1). This suggests that if the B ring and the gallate group of (+)-EGCGwere to bind in the same position in three-dimensional space as(−)-EGCG, the A-C rings of (+)-EGCG would then have to rotate 180degrees to compensate (FIG. 3B-1). Thus, the proteasome does not exhibitsignificant enantioselectivity for EGCG (FIG. 3) due to the partialsymmetry of the A-C rings.

(−)-GCG is a non-“epi” compound that has trans stereochemistry about theC ring unlike (−)-EGCG which has cis stereochemistry (FIG. 1). The IC₅₀value of (−)-GCG indicates that it is nearly 3 times less potent then(−)-EGCG (610 nM vs. 205 nM; FIGS. 3A and 3C), suggesting that the transstereochemistry may not be as beneficial for binding to the proteasome'sactive site. In agreement with the experimental IC₅₀ values, thecalculated binding free energy of (−)-GCG was −9.10 kcal/mol (FIG. 3C)compared with −10.52 kcal/mol for (−)-EGCG (FIG. 3A). For clarity, atwo-dimensional scheme of the binding mode for (−)-GCG is alsorepresented (FIG. 3G).

The synthetic (+)-GCG was more potent than the natural (−)-GCG (270 nMvs. 610 nM; FIGS. 3C and 3D). Consistent with their IC₅₀ values, a lowerfree energy is required for binding of (+)-GCG to β5 subunit than thatof (−)-GCG (−10.33 kcal/mol vs. −9.10 kcal/mol; FIGS. 3C and 3D).(+)-GCG binds in a slightly different conformation compared with therest of the other compounds (FIG. 3H). The unique (+)-transstereochemistry of (+)-GCG allows for its B ring to form three H-bondswith Thr 21, Gly 23, and Tyr 170 (FIGS. 3D and 3H) instead of two aswith (−)-EGCG (FIG. 2E). It also hydrophobically interacts with Tyr 170(see FIG. 3H), which has stronger affinity than the binding cleftbridging conformation. The gallate group of (+)-GCG also extends furtherout of the pocket and forms three H-bonds with residues Val 129, Gly 130and Ser 131 (FIGS. 3D and 3H), instead of two H-bonds as with (−)-EGCG(FIG. 2D). However, while this conformation may increase bindingaffinities at the B ring and gallate moieties, the A-C rings are pulledslightly out of the S1 pocket, reducing the total number of interactionsthat take place there. As a net result, a slight overall reduction inbinding free energy and a slight reduction in in vitroproteasome-inhibitory activity occurs, as compared to (−)-EGCG (FIGS. 3Dvs. 3A).

The natural GTP (−)-ECG lacks one hydroxyl group on its B ring (FIG. 1),which significantly reduces its solubility in water and also decreasesits potency against 20S proteasome by more than three-fold, as comparedto (−)-EGCG (710 nM vs. 205 nM; FIGS. 3A and 3E). (−)-ECG is also foundto bind the β5 binding cleft with almost exactly the same binding modeas (−)-EGCG except for the loss of the H bond with the side chain of Thr21 (FIGS. 3E vs. 2E). However, this did not increase the calculatedbinding free energy (−10.56 vs. −10.52 kcal/mol; FIG. 3E). Because the Bring is protruding into solvent and, as mentioned previously, the lossof this hydroxyl significantly decreases the solubility of (−)-ECG,Binding of this GTP to the proteasome might be affected in a manner thatis not well accounted for by the solvation model used in the dockingalgorithms.

The natural GTP (−)-CG is another non-epi compound with a transstereochemistry (FIG. 1) and is less potent than (−)-EGCG (FIGS. 3F vs.3A). Consistent with this, an increased ligand free energy is calculatedfor binding of (−)-CG to the proteasome's active site, thereby giving anet increase in binding free energy [−9.30 kcal/mol vs. −10.52 kcal/mol;and see the discussion about (−)-GCG].

Genistein, the predominant isoflavone found in soy products, wasselected to test whether the developed computational model can beapplied to a range of compounds with different chemical structures. Like(−)-EGCG, genistein also consists of a ring system similar to the A, C,and B rings of the GTPs (see FIG. 1), suggesting that genistein might bea proteasome inhibitor. But different from (−)-EGCG, genistein lacks thegallate group (see FIG. 1), which suggests that genistein would be lesspotent than (−)-EGCG.

Genistein was docked to yeast 20S proteasome. In 60 out of 100 runs with5-million energy evaluations, genistein docks primarily in the S1 pocketof the active site of the proteasome β5 subunit. The B ring hydroxylgroup of genistein lies in close proximity to Thr 1, and there are fourpotential hydrogen bonds that could be formed within the complex ofgenistein and the proteasomal β5 subunit. However, the binding freeenergy of genistein to β5 subunit was found to be −5.15 kcal/mol, muchhigher than that of (−)-EGCG (−10.52 kcal/mol; FIG. 3A). Consistent withits higher docking energy, genistein weakly inhibits thechymotrypsin-like activity of purified 20S proteasome with an IC₅₀ valueof 26 μM (also see Table 1 and FIG. 5), in contrast to an IC₅₀ of 205 nMfor (−)-EGCG (FIG. 3A). These data further demonstrates establishedcomputational model satisfactorily describes the EGCG-β5 interactionthat is responsible for its proteasome-inhibitory activity.

Finally, to compare the selected binding modes to the actualproteasome-inhibitory activities of each of the eight EGCG analogs andgenistein, the predicted activity (binding-free energy) against theactual inhibitory activity (IC₅₀ values; converted to kcal/mol) (Table 1and FIG. 5). A decrease in the docking free energy for 8 of the 9compounds was correlated with an increase in the actual activity of eachof these compounds. Only one compound, (−)-ECG, did not fit the linearrelationship between the predicted and actual activity (FIG. 5). Thissignificant loss in actual activity of (−)-ECG, which is not incongruence with the calculated binding free energy, may be due to theorientation and solvation issues mentioned previously. A regressionanalysis R² value of 0.9893 was determined for a best-fit line, notincluding the values generated for (−)-ECG.

TABLE 1 Predicted vs. Observed Binding Free Energies Compound PredictedΔG° (kcal/mol) IC₅₀ −RTln(l/IC₅₀)^(a) (+)-EGCG −10.82 170 nM −9.60(−)-EGCG −10.52 205 nM −9.49 (+)-GCG −10.33 270 nM −9.32 (−)-ECG −10.56710 nM −8.72 (−)-EGCG-Amide −9.63 320 nM −9.21 (+)-EGCG-Amide −9.52 405nM −9.07 (−)-CG −9.30 505 nM −8.93 (−)-GCG −9.10 610 nM −8.81 genistein−5.15  26 μM −6.50 ^(a)Note that IC₅₀ is proportional to K_(i). SinceK_(i) is the equilibrium constant for the dissociation of theenzyme-inhibitor complex, and the binding free energy (ΔG°) is relatedto the equilibrium constant for the association of enzyme withinhibitor, ΔG° is proportional to −RTln(l/IC₅₀), which is identical to+RTln(IC₅₀).

Example 11 (−)-EGCG Proteasome Inhibition

To investigate the nature of (−)-EGCG-mediated proteasome inhibition, adialysis experiment was performed. A purified prokaryotic 20S proteasomewas pre-incubated for one hour at 37° C. with either 10 μM (−)-EGCG orits control solvent (H₂O), followed by overnight co-incubation at 4° C.with or without dialysis.

FIG. 6 shows that, in the absence of dialysis, (−)-EGCG was able toinhibit the chymotrypsin-like activity of the prokaryotic 20S proteasomeby 85%. More importantly, overnight dialysis of the EGCG-proteasomemixture did not change the outcome: 1 h pre-incubation of (−)-EGCG stillcaused 81% inhibition of the proteasomal chymotrypsin-like activity.

As a control, when an aliquot of (−)-EGCG was first dialyzed overnightand then added to the purified 20S proteasome, no inhibition wasobserved (FIG. 6, EGCG pre). This result demonstrates that (−)-EGCG iseither an irreversible or a tight-binding inhibitor of thechymotrypsin-like activity of the proteasome.

In the second experiment (−)-EGCG at 1 μM was pre-incubated with(SDS-pre) or without 0.01% SDS (no SDS) for 1 hour, followed by additionof eukaryotic 20S proteasome (0.02 μg) and suc-LLVY-AMC (20 μM).“SDS-post” represents the same treatment as “no SDS” except 0.01% SDSwas added 1 hour after addition of the proteasome, (−)-EGCG and thesubstrate. AMC liberation was measured by fluorescence at each indicatedtime point and the percentage of chymotrypsin activity was determined.Values are means from 4 independent experiments, and error barsrepresent standard deviations.

In the second experiment, (−)-EGCG at 1 μM potently inhibited thechymotrypsin-like activity of a purified eukaryotic (rabbit) 20Sproteasome (up to 75%; FIG. 22B, no SDS). However, pre-incubation with0.01% SDS for 1 hour resulted in a significant loss of the potency of(−)-EGCG (to 10-20% of inhibition; FIG. 22B, SDS-pre), suggesting thatSDS antagonizes EGCG's ability to inhibit the proteasome. When (−)-EGCGwas pre-incubated with the 20S proteasome for 1 hour, addition of SDScould not reverse the EGCG-mediated proteasome inhibition (FIG. 22B,SDS-post), demonstrating that EGCG had already inhibited the proteasomeafter 1 hour and this inhibition could not be antagonized by SDS. In theabsence of (−)-EGCG, 0.01% SDS was able to increase the 20S proteasomalactivity.

The result of the second experiment demonstrates that the mode of(−)-EGCG action is an irreversible inhibition by a covalent bondformation, not tight binding inhibition. Furthermore, the time-dependentinhibition of the proteasomal chymotrypsin-like activity by (−)-EGCG:35% at 5 min, 62% at 30 min, and 70-80% after 1 to 3 hours (FIG. 22B, noSDS) is characteristic for a mechanism-based inhibitor.

Kinetic analysis and X-ray diffraction studies using the specificproteasome inhibitor lactacystin have demonstrated that the ester bondof the inhibitor covalently modifies the N-terminal threonine of the β5subunit, which is critical for proteasome inhibition. Since (−)-EGCGcontains an ester bond (FIG. 15) and inhibits the proteasomeirreversibly in a time-dependent manner (FIGS. 22A and 22B), it isprobable that a lactacystin-like reaction could occur with (−)-EGCG.

The prokaryotic proteasome was used because it demonstrated more stablekinetics after overnight dialysis, although a similar result can beobtained using eukaryotic proteasome.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

1. A compound having a formula of:

wherein R₁-R₉ are independently selected from the group consisting of—H, alkyl, alkenyl, cycloalkyl, heterocycloalkyl, cycloalkenyl,heterocycloalkenyl, aryl, and acyl; or a composition comprising saidcompound, or a pharmaceutically acceptable salt or analog thereof; and apharmaceutically acceptable carrier or diluent.
 2. The compoundaccording to claim 1, wherein said compound is (−)-EGCG-amide or(+)-EGCG-amide.
 3. A method for inhibiting proteasomal activity in cellscomprising: a) contacting the cells with an effective amount of at leastone compound of claim 1, or a pharmaceutically acceptable salt or analogthereof; and b) verifying that proteasomal activity has been inhibited.4. The method according to claim 3, wherein said compound is(+)-EGCG-amide or (−)-EGCG-amide, or a pharmaceutically acceptable saltor analog thereof.
 5. The method according to claim 3, wherein saidcontacting occurs in vivo, and said contacting comprises administeringthe compound to a patient by a route selected from the group consistingof orally, parenterally, subcutaneously, intravenously, intramuscularly,intraperitoneally, intraarterially, transdermally, and via a mucusmembrane.
 6. The method according to claim 3, wherein said contactingoccurs in vitro.
 7. The method according to claim 3, wherein saidcompound has less than 100% optical purity.
 8. The method according toclaim 3, wherein said compound is optically pure.
 9. The methodaccording to claim 3, wherein said proteasome is a 20S proteasome or 26Sproteasome.
 10. A method for treating cancer comprising administering aneffective amount of a compound or composition of claim 1 to a patient.11. The method according to claim 10, wherein said cancer is selectedfrom the group consisting of prostate cancer, leukemia, hormonedependent cancers, breast cancer, colon cancer, lung cancer, epidermalcancer, liver cancer, esophageal cancer, stomach cancer, cancer of thebrain, and cancer of the kidney.
 12. The method according to claim 10,wherein said administering is conducted by a route selected from thegroup consisting of orally, parenterally, subcutaneously, intravenously,intramuscularly, intraperitoneally, intraarterially, transdermally, andvia a mucus membrane.
 13. The method according to claim 10, wherein saidpharmaceutical composition comprises (−)-EGCG-amide; or (+)-EGCG-amide;or a pharmaceutically acceptable salt or analog thereof; or a mixturethereof.
 14. A method for synthesizing phenols, said method comprising:a) coupling a compound represented by formula II with an acidrepresented by formula III:

to form a fully protected gallate ester, wherein R₁-R₉ are independentlyselected from the group consisting of —H, alkyl, acyl, alkenyl,cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkyl, and aryl.15. The method according to claim 14, wherein the acid of formula III isemployed in the form of a derivative which is an acyl halide or a mixedor symmetric acid anhydride; or the acid of formula III is reacted withthe compound of formula II in the presence of a condensing reagent. 16.A compound produced from a method comprising: a) coupling a compound offormula II with an acid of formula III:

to form a fully protected gallate ester, wherein R₁-R₉ are independentlyselected from the group consisting of —H, alkyl, acyl, cycicoalkyl,heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, and aryl; whereinsaid coupling is carried out in the presence of a base in an inertsolvent; and b) deprotecting the gallate ester, wherein saiddeprotecting is selective or non-selective.
 17. A method of a)increasing the relative proportion of cells occupying the G₁ phase ofthe mitotic cell cycle within a plurality of cells; or b) inducingapoptosis in a plurality of cells; said method comprising contacting theplurality of cells with an effective amount of a compound having aformula of:

wherein R₁-R₉ are independently selected from the group consisting of—H, alkyl, alkenyl, cycloalkyl, heterocycloalkyl, cycloalkenyl,heterocycloalkenyl, aryl, and acyl; or a composition comprising saidcompound, or a pharmaceutically acceptable salt or analog thereof; and apharmaceutically acceptable carrier or diluent.
 18. The method accordingto claim 17, wherein said cells comprise cancer cells.